Varicella zoster virus (vzv) vaccine

ABSTRACT

Aspects of the disclosure relate to nucleic acid vaccines. The vaccines include at least one RNA polynucleotides having an open reading frame encoding at least one varicella zoster virus (VZV) antigen. Methods for preparing and using such vaccines are also described.

RELATED APPLICATIONS

This application claims the benefit under 35 U.S.C. § 119(e) of U.S.provisional application No. 62/471,809, filed Mar. 15, 2017, and U.S.provisional application No. 62/490,112, filed Apr. 26, 2017, each ofwhich is incorporated by reference herein in its entirety.

BACKGROUND

Varicella is an acute infectious disease caused by varicella zostervirus (VZV). Varicella zoster virus is one of eight herpesviruses knownto infect humans and vertebrates. VZV is also known as chickenpox virus,varicella virus, zoster virus, and human herpesvirus type 3 (HHV-3). VZVonly affects humans, and commonly causes chickenpox in children, teensand young adults and herpes zoster (shingles) in adults (rarely inchildren). The primary VZV infection, which results in chickenpox(varicella), may result in complications, including viral or secondarybacterial pneumonia. Even when the clinical symptoms of chickenpox haveresolved, VZV remains dormant in the nervous system of the infectedperson (virus latency) in the trigeminal and dorsal root ganglia. Inabout 10-20% of cases, VZV reactivates later in life, travelling fromthe sensory ganglia back to the skin where it produces a disease (rash)known as shingles or herpes zoster. VZV can also cause a number ofneurologic conditions ranging from aseptic meningitis to encephalitis.Other serious complications of VZV infection include postherpeticneuralgia, Mollaret's meningitis, zoster multiplex, thrombocytopenia,myocarditis, arthritis, and inflammation of arteries in the brainleading to stroke, myelitis, herpes ophthalmicus, or zoster sineherpete. In rare instances, VZV affects the geniculate ganglion, givinglesions that follow specific branches of the facial nerve. Symptoms mayinclude painful blisters on the tongue and ear along with one sidedfacial weakness and hearing loss.

Varicella cases have declined 97% since 1995, mostly due to vaccination.However, an estimated 500,000 to 1 million episodes of herpes zoster(shingles) occur annually in just the United States. The lifetime riskof herpes zoster is estimated to be at least 32%, with increasing ageand cellular immunosuppression being the most important risk factors. Infact, it is estimated that 50% of persons living until the age of 85will develop herpes zoster.

A live attenuated VZV Oka strain vaccine is available and is marketed inthe United States under the trade name VARIVAX® (Merck). A similar, butnot identical, VZV vaccine is marketed globally as VARILRIX®(GlaxoSmithKline). Since its approval in 1995, it has been added to therecommended vaccination schedules for children in Australia, the UnitedStates, and several other countries. In 2007, the Advisory Committee onImmunization Practices (ACIP) recommended a second dose of vaccinebefore school entry to ensure the maintenance of high levels ofvaricella immunity. In 2001-2005, outbreaks were reported in schoolswith high varicella vaccination coverage, indicating that even insettings where most children were vaccinated and the vaccine performedas expected, varicella outbreaks could not be prevented with theone-dose vaccination policy. As a result, two-dose vaccination is theadopted protocol; however, even with two doses of vaccine, there arereported incidences of breakthrough varicella. Furthermore, varicellavaccination has raised concerns that the immunity induced by the vaccinemay not be lifelong, possibly leaving adults vulnerable to more severedisease as the immunity from their childhood immunization wanes.

In 2005, the FDA approved the combined live attenuated combinationmeasles-mumps-rubella-varicella (MMRV) vaccine PROQUAD™ (Merck) for usein persons 12 months to 12 years in age. While the attenuated measles,mumps, and rubella vaccine viruses in MMRV are identical and of equaltiter to those in the MMR vaccine, the titer of Oka/Merck VZV is higherin MMRV vaccine than in single-antigen varicella vaccine.

In 2006, the United States Food and Drug Administration approvedZOSTAVAX® (Merck) for the prevention of shingles (herpes zoster) inpersons 60 years or older (currently 50-59 years of age is approved).ZOSTAVAX® contains the same Oka/Merck varicella zoster virus used in thevaricella and MMRV vaccines, but at a much higher titer (>10-fold higherviral dose) than that present in both of these vaccines, as theconcentrated formulation is designed to elicit an immune response inolder adults whose immunity to VZV wanes with advancing age.

Although the varicella vaccine has been shown to be safe in healthyindividuals, there is evidence that immunity to VZV infection conferredby the vaccine wanes over time, rendering the vaccinated individualssusceptible to shingles, a more serious condition. In addition, therehave been reports that individuals have developed chicken pox orshingles from the varicella vaccination. The vaccine may establish alatent infection in neural ganglia, which can then reactivate to causeherpes zoster.

Moreover, live attenuated virus is not suitable for all subjects,including pregnant women and persons with moderate or severe acuteillnesses. Also, varicella is not suitable or approved forimmunocompromised patients, including persons with immunosuppression dueto leukemia, lymphoma, generalized malignancy, immune deficiency diseaseor immunosuppressive therapy. Likewise, persons with moderate or severecellular immunodeficiency resulting from infection with humanimmunodeficiency virus (HIV) including those diagnosed with acquiredimmunodeficiency syndrome (AIDS) should not receive the varicellavaccine. Thus, despite the high risk of morbidity and mortalityassociated with herpes zoster in immunocompromised individuals, thispopulation is not eligible for vaccination with a live attenuatedvaccine, such as ZOSTAVAX®.

There are one million cases of herpes zoster in the U.S. each year. Anestimated $1 billion is spent annually on direct medical costs forherpes zoster in the US and treatment for herpes zoster is not alwayseffective or available.

Deoxyribonucleic acid (DNA) vaccination is one technique used tostimulate humoral and cellular immune responses to foreign antigens,such as VZV antigens. The direct injection of genetically engineered DNA(e.g., naked plasmid DNA) into a living host results in a small numberof host cells directly producing an antigen, resulting in a protectiveimmunological response. With this technique, however, comes potentialproblems, including the possibility of insertional mutagenesis, whichcould lead to the activation of oncogenes or the inhibition of tumorsuppressor genes.

SUMMARY

Provided herein is a ribonucleic acid (RNA) vaccine that builds on theknowledge that RNA (e.g., messenger RNA (mRNA)) can safely direct thebody's cellular machinery to produce nearly any protein of interest,from native proteins to antibodies and other entirely novel proteinconstructs that can have therapeutic activity inside and outside ofcells. The varicella zoster virus (VZV) RNA vaccines of the presentdisclosure may be used to induce a balanced immune response against VZVcomprising both cellular and humoral immunity, without many of the risksassociated with attenuated virus vaccination.

The RNA (e.g., mRNA) vaccines may be utilized in various settingsdepending on the prevalence of the infection or the degree or level ofunmet medical need. The RNA (e.g., mRNA) vaccines may be utilized totreat and/or prevent a VZV of various genotypes, strains, and isolates.The RNA (e.g., mRNA) vaccines have superior properties in that theyproduce much larger antibody titers and produce responses earlier thancommercially available anti-viral therapeutic treatments. While notwishing to be bound by theory, it is believed that the RNA vaccines, asmRNA polynucleotides, are better designed to produce the appropriateprotein conformation upon translation as the RNA vaccines co-opt naturalcellular machinery. Unlike traditional vaccines, which are manufacturedex vivo and may trigger unwanted cellular responses, RNA (e.g., mRNA)vaccines are presented to the cellular system in a more native fashion.

Various VZV amino acid sequences encompasses by the present disclosureare provided in Tables 1-9. RNA (e.g., mRNA) vaccines as provided hereinmay include at least one RNA polynucleotide encoding at least one of theVZV glycoproteins provided in Table 1, or a fragment, homolog (e.g.,having at least 80%, 85%, 90%, 95%, 98% or 99% identity) or variant orderivative thereof.

Some embodiments of the present disclosure provide VZV vaccines thatinclude at least one ribonucleic acid (RNA) polynucleotide having anopen reading frame encoding at least one VZV antigenic polypeptide. Someembodiments of the present disclosure provide VZV vaccines that includeat least one RNA polynucleotide having an open reading frame encodingtwo or more VZV antigenic polypeptides. Some embodiments of the presentdisclosure provide VZV vaccines that include two or more RNApolynucleotides having an open reading frame encoding two or more VZVantigenic polypeptides.

In some embodiments, an antigenic polypeptide is a VZV glycoprotein. Forexample, a VZV glycoprotein may be VZV gE, gI, gB, gH, gK, gL, gC, gN,or gM. In some embodiments, the antigenic polypeptide is a VZV gEpolypeptide. In some embodiments, the antigenic polypeptide is a VZV gIpolypeptide. In some embodiments, the antigenic polypeptide is a VZV gBpolypeptide. In some embodiments, the antigenic polypeptide is a VZV gHpolypeptide. In some embodiments, the antigenic polypeptide is a VZV gKpolypeptide. In some embodiments, the antigenic polypeptide is a VZV gLpolypeptide. In some embodiments, the antigenic polypeptide is a VZV gCpolypeptide. In some embodiments, the antigenic polypeptide is a VZV gNpolypeptide. In some embodiments, the antigenic polypeptide is a VZV gMpolypeptide. In some embodiments, the VZV glycoprotein is encoded by anucleic acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

In some embodiments, the VZV glycoprotein is a variant gE polypeptide.In some embodiments, the variant VZV gE polypeptide is a truncatedpolypeptide lacking the anchor domain (ER retention domain). In someembodiments, the truncated VZV gE polypeptide comprises (or consists of,or consists essentially of) amino acids 1-561 of VZV gE polypeptide. Insome embodiments, the truncated VZV gE polypeptide comprises (orconsists of, or consists essentially of) amino acids 1-561 of SEQ ID NO:10. In some embodiments, the truncated VZV gE polypeptide comprises (orconsists of, or consists essentially of) amino acids 1-573 of SEQ ID NO:18. In some embodiments, the truncated VZV gE polypeptide comprises (orconsists of, or consists essentially of) amino acids 1-573 of SEQ ID NO:10. In some embodiments, the variant VZV gE polypeptide is a truncatedpolypeptide lacking the carboxy terminal tail domain. In someembodiments, the truncated VZV gE polypeptide comprises (or consists of,or consists essentially of) amino acids 1-573 of VZV gE polypeptide. Insome embodiments, the truncated VZV gE polypeptide comprises (orconsists of, or consists essentially of) amino acids 1-573 of SEQ ID NO:34.

In some embodiments, the variant VZV gE polypeptide has at least onemutation in one or more motif(s) associated with ER retention, whereinthe mutation(s) in one or more motif(s) results in decreased retentionof the VZV gE polypeptide in the ER and/or golgi. In some embodiments,the variant VZV gE polypeptide has at least one mutation in one or moremotif(s) associated with targeting gE to the golgi or trans-golginetwork (TGN), wherein the mutation(s) in one or more motif(s) resultsin decreased targeting or localization of the VZV gE polypeptide to thegolgi or TGN. In some embodiments, the variant VZV gE polypeptide has atleast one mutation in one or more motif(s) associated with theinternalization of VZV gE or the endocytosis of gE, wherein themutation(s) in one or more motif(s) results in decreased endocytosis ofthe VZV gE polypeptide. In some embodiments, the variant VZV gEpolypeptide has at least one mutation in one or more phosphorylatedacidic motif(s), such as SSTT (SEQ ID NO: 122). In some embodiments, thevariant VZV gE polypeptide is a full-length VZV gE polypeptide having aY582G mutation. In some embodiments, the variant VZV gE polypeptide is afull-length VZV gE polypeptide having a Y569A mutation. In someembodiments, the variant VZV gE polypeptide is a full-length VZV gEpolypeptide having a Y582G mutation and a Y569A mutation. In someembodiments, the variant VZV gE polypeptide is an antigenic fragmentcomprising amino acids 1-573 of VZV gE and having a Y569A mutation. Insome embodiments, the variant VZV gE polypeptide is an antigenicfragment comprising SEQ ID NO: 38.

In some embodiments, the variant VZV gE polypeptide is a full-length VZVgE polypeptide having an IgK sequence. In some embodiments, the variantVZV gE polypeptide is SEQ ID NO: 14. In some embodiments, the variantVZV gE polypeptide is a full-length VZV gE polypeptide having anA-E-A-A-D-A sequence (SEQ ID NO: 58) that replaces SESTDT (SEQ ID NO:59). This is a replacement of the Ser/Thr-rich “SSTT” (SEQ ID NO: 122)acidic cluster with an Ala-rich sequence. In some embodiments, thevariant VZV gE polypeptide is SEQ ID NO: 26. In some embodiments inwhich the VZV gE polypeptide has an A-E-A-A-D-A sequence (SEQ ID NO:58), the variant VZV gE polypeptide also has at least one mutation inone or more motif(s) associated with ER/golgi retention, TGNlocalization, or endocytosis (e.g., has a Y582G mutation, a Y569Amutation, or both a Y582G mutation and a Y569A mutation) and/or has atleast one mutation in one or more phosphorylated acidic motif(s), suchas a SSTT (SEQ ID NO: 122) motif. In some embodiments, the variant VZVgE polypeptide is or comprises the amino acid sequence of SEQ ID NO: 30.

In some embodiments, the variant VZV gE polypeptide is a full-length VZVgE polypeptide having an additional sequence at the C-terminus that aidsin secretion of the polypeptide or its localization to the cellmembrane. In some embodiments, the variant VZV gE polypeptide is afull-length VZV gE polypeptide having an IgKappa sequence at theC-terminus. In some embodiments, the VZV gE polypeptide has additionalsequence at the C-terminus that aids in secretion (e.g., has an IgKappasequence at the C-terminus) and has at least one mutation in one or moremotif(s) associated with ER retention, TGN localization, or endocytosis(e.g., has a Y582G mutation, a Y569A mutation, or both a Y582G mutationand a Y569A mutation) and/or has at least one mutation in one or morephosphorylated acidic motif(s), such as the SSTT (SEQ ID NO: 122) motif.In some embodiments, the variant VZV gE polypeptide is a truncatedpolypeptide lacking the anchor domain (ER retention domain) and havingan additional sequence at the C-terminus that aids in secretion of thepolypeptide (e.g., an IgKappa sequence at the C-terminus). In someembodiments, the truncated VZV gE polypeptide comprises amino acids1-561 and has an IgKappa sequence at the C-terminus. In someembodiments, the variant polypeptide is SEQ ID NO: 22. In someembodiments, the variant VZV gE polypeptide is a truncated polypeptidelacking the carboxy terminal tail domain and having an additionalsequence at the C-terminus that aids in secretion of the polypeptide,for example, having an IgKappa sequence at the C-terminus. In someembodiments, the truncated VZV gE polypeptide comprises amino acids1-573 and has an IgKappa sequence at the C-terminus.

In some embodiments, the antigenic polypeptide comprises two or moreglycoproteins. In some embodiments, the two or more glycoproteins areencoded by a single RNA polynucleotide. In some embodiments, the two ormore glycoproteins are encoded by two or more RNA polynucleotides, forexample, each glycoprotein is encoded by a separate RNA polynucleotide.In some embodiments, the two or more glycoproteins can be anycombination of VZV gE, gI, gB, gH, gK, gL, gC, gN, and gM polypeptides.In some embodiments, the two or more glycoproteins can be anycombination of VZV gE and at least one of gI, gB, gH, gK, gL, gC, gN,and gM polypeptides. In some embodiments, the two or more glycoproteinscan be any combination of VZV gI and at least one of gE, gB, gH, gK, gL,gC, gN, and gM polypeptides. In some embodiments, the two or moreglycoproteins can be any combination of VZV gE, gI, and at least one ofgB, gH, gK, gL, gC, gN, and gM polypeptides.

In some embodiments, the two or more VZV glycoproteins are gE and gI. Insome embodiments, the two or more VZV glycoproteins are gE and gB. Insome embodiments, the two or more VZV glycoproteins are gI and gB. Insome embodiments, the two or more VZV glycoproteins are gE, gI, and gB.In some embodiments, the two or more VZV glycoproteins are gE and gH. Insome embodiments, the two or more VZV glycoproteins are gI and gH. Insome embodiments, the two or more VZV glycoproteins are gE, gI, and gH.In some embodiments, the two or more VZV glycoproteins are gE and gK. Insome embodiments, the two or more VZV glycoproteins are gI and gK. Insome embodiments, the two or more VZV glycoproteins are gE, gI, and gK.In some embodiments, the two or more VZV glycoproteins are gE and gL. Insome embodiments, the two or more VZV glycoproteins are gI and gL. Insome embodiments, the two or more VZV glycoproteins are gE, gI, and gL.In some embodiments, the two or more VZV glycoproteins are gE and gC. Insome embodiments, the two or more VZV glycoproteins are gI and gC. Insome embodiments, the two or more VZV glycoproteins are gE, gI, and gC.In some embodiments, the two or more VZV glycoproteins are gE and gN. Insome embodiments, the two or more VZV glycoproteins are gI and gN. Insome embodiments, the two or more VZV glycoproteins are gE, gI, and gN.In some embodiments, the two or more VZV glycoproteins are gE and gM. Insome embodiments, the two or more VZV glycoproteins are gI and gM. Insome embodiments, the two or more VZV glycoproteins are gE, gI, and gM.

In some embodiments, the vaccine comprises any two or more VZVglycoproteins (e.g., any of the variant VZV gE disclosed in thepreceding paragraphs and in the Examples and Figures), and the VZV gE isa variant gE, such as any of the variant VZV gE glycoproteins disclosedherein, for example, any of the variant VZV gE disclosed in thepreceding paragraphs and in the Examples and Figures.

In some embodiments, the VZV vaccine includes two or more RNApolynucleotides having an open reading frame encoding two or more VZVantigenic polypeptides (either encoded by a single RNA polynucleotide orencoded by two or more RNA polynucleotides, for example, eachglycoprotein encoded by a separate RNA polynucleotide), and the two ormore VZV glycoproteins are a variant gE (e.g., any of the variant gEpolypeptides disclosed herein in the preceding paragraphs) and a VZVglycoprotein selected from gI, gB, gH, gK, gL, gC, gN, and gMpolypeptides. In some embodiments, the two or more VZV glycoproteins area variant gE (e.g., any of the variant gE polypeptides disclosed hereinin the preceding paragraphs) and gI. In some embodiments, theglycoproteins are VZV gI and variant VZV gE, and the variant VZV gEpolypeptide is a truncated polypeptide lacking the anchor domain (ERretention domain) (e.g., a truncated VZV gE polypeptide comprising aminoacids 1-561 of SEQ ID NO: 10). In some embodiments, the glycoproteinsare VZV gI and variant VZV gE, and the variant VZV gE polypeptide is atruncated polypeptide lacking the carboxy terminal tail domain (e.g., atruncated VZV gE polypeptide comprising amino acids 1-573 of SEQ ID NO:18). In some embodiments, the glycoproteins are VZV gI and variant VZVgE, and the variant VZV gE polypeptide has at least one mutation in oneor more motif(s) associated with ER retention, TGN localization, and/orendocytosis (e.g., the variant VZV gE has a Y582G mutation, a Y569Amutation, or both a Y582G mutation and a Y569A mutation) and/or has atleast one mutation in one or more phosphorylated acidic motif(s), suchas SSTT (SEQ ID NO: 122) motif. In some embodiments, the glycoproteinsare VZV gI and variant VZV gE, and the variant VZV gE polypeptide is anantigenic fragment comprising amino acids 1-573 of VZV gE and having aY569A mutation. In some embodiments, the glycoproteins are VZV gI andvariant VZV gE, and the variant VZV gE polypeptide is a full-length VZVgE polypeptide having an A-E-A-A-D-A (SEQ ID NO: 58) sequence. In someembodiments, the glycoproteins are VZV gI and variant VZV gE, and theVZV gE polypeptide has an A-E-A-A-D-A (SEQ ID NO: 58) sequence and aY582G mutation, a Y569A mutation, or both a Y582G mutation and a Y569Amutation. In some embodiments, the glycoproteins are VZV gI and variantVZV gE, and the VZV gE polypeptide is a full-length VZV gE polypeptidehaving an additional sequence at the C-terminus that aids in secretionof the polypeptide (e.g., an IgKappa sequence). In some embodiments, theglycoproteins are VZV gI and variant VZV gE, and the VZV gE polypeptideis a full-length VZV gE polypeptide having an IgKappa sequence and aY582G mutation, a Y569A mutation, or both a Y582G mutation and a Y569Amutation. In some embodiments, the glycoproteins are VZV gI and variantVZV gE, and the VZV gE polypeptide is a truncated VZV gE polypeptidelacking the anchor domain (ER retention domain) and having an IgKappasequence. In some embodiments, the variant VZV gE polypeptide is atruncated polypeptide comprising amino acids 1-561 or amino acids 1-573and having an IgKappa sequence at the C-terminus.

In any of the above-described embodiments, the VZV vaccine may furthercomprise a live attenuated VZV, a whole inactivated VZV, or a VZVvirus-like particle (VLP). In some embodiments, the live attenuated VZV,whole inactivated VZV, or VZV VLP is selected from or derived from thefollowing strains and genotypes: VZV E1 strain, genotypes E1_32_5,E1_Kel, E1_Dumas, E1_Russia 1999, E1_SD, E1_MSP, E1_36, E1_49, E1_BC,E1_NH29; VZV E2 strain, genotypes E2_03-500, E2_2, E2_11, E2_HJO; VZV Jstrain, genotype pOka; VZV M1 strain, genotype M1_CA123; VZV M2 strain,genotypes M2_8 and M2_DR; and VZV M4 strain, genotypes Spain 4242,France 4415, and Italy 4053.

Alternate RNA vaccines comprising RNA polynucleotides encoding otherviral protein components of VZV, for example, tegument proteins areencompassed by the present disclosure. Thus, some embodiments of thepresent disclosure provide VZV vaccines that include at least oneribonucleic acid (RNA) polynucleotide having an open reading frameencoding at least one VZV tegument protein. Some embodiments of thepresent disclosure provide VZV vaccines that include at least one RNApolynucleotide having an open reading frame encoding at least one VZVtegument protein and at least one VZV glycoprotein. Some embodiments ofthe present disclosure provide VZV vaccines that include at least oneRNA polynucleotide having an open reading frame encoding at least oneVZV tegument protein and at least one RNA polynucleotide having an openreading frame encoding at least one VZV glycoprotein. In someembodiments, RNA vaccines comprise RNA (e.g., mRNA) polynucleotide(s)encoding one or more VZV tegument protein(s) and one or more VZVglycoprotein(s) selected from VZV gE, gI, gB, gH, gK, gL, gC, gN, and gMpolypeptides. In some embodiments, the VZV glycoprotein is a VZV gEpolypeptide. In some embodiments, the VZV glycoprotein is a VZV gIpolypeptide. In some embodiments, the VZV glycoprotein is a variant VZVgE polypeptide, such as any of the variant VZV gE polypeptides disclosedherein. In some embodiments, the VZV glycoproteins are VZV gEglycoproteins and VZV gI glycoproteins.

In some embodiments, at least one RNA polynucleotide is encoded by atleast one nucleic acid sequence selected from SEQ ID NO: 1, SEQ ID NO:2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7,SEQ ID NO: 8, SEQ ID NO: 41 and homologs having at least 80% (e.g., 85%,90%, 95%, 98%, 99%) identity with a nucleic acid sequence selected fromSEQ ID NO: 1-8 and 41. In some embodiments, at least one RNApolynucleotide is encoded by at least one nucleic acid sequence selectedfrom SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO:5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 41 and homologshaving at least 90% (e.g., 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99%, 99.8% or 99.9%) identity with a nucleic acid sequence selected fromSEQ ID NO: 1-8 and 41. In some embodiments, at least one RNApolynucleotide is encoded by at least one fragment of a nucleic acidsequence (e.g., a fragment having an antigenic sequence or at least oneepitope) selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ IDNO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, SEQ IDNO: 41 and homologs having at least 80% (e.g., 85%, 90%, 95%, 98%, 99%)identity with a nucleic acid sequence selected from SEQ ID NO: 1-8 and41. In some embodiments, at least one RNA polynucleotide is encoded byat least one epitope of a nucleic acid sequence selected from SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6,SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 41.

In some embodiments, at least one RNA polynucleotide is a gE polypeptideencoded by SEQ ID NO: 1. In some embodiments, at least one RNApolynucleotide is a gI polypeptide encoded by SEQ ID NO: 2. In someembodiments, at least one RNA polynucleotide is a truncated gEpolypeptide encoded by SEQ ID NO: 3. In some embodiments, at least oneRNA polynucleotide is a truncated gE polypeptide encoded by SEQ ID NO:5. In some embodiments, at least one RNA polynucleotide is a truncatedgE polypeptide having Y569A mutation encoded by SEQ ID NO: 6. In someembodiments, at least one RNA polynucleotide is a gE polypeptide havingan AEAADA sequence SEQ ID NO: 58 encoded by SEQ ID NO: 7. In someembodiments, at least one RNA polynucleotide is a gE polypeptide havinga Y582G mutation and a AEAADA sequence (SEQ ID NO: 58) encoded by SEQ IDNO: 8. In some embodiments, at least one RNA polynucleotide is a gEpolypeptide encoded by SEQ ID NO: 41.

In some embodiments, at least one RNA (e.g., mRNA) polynucleotideencodes an antigenic polypeptide having at least 90% identity to theamino acid sequence of any one of SEQ ID NO: 10, 14, 18, 22, 26, 30, 34,38, 42 and 45-55. In some embodiments, at least one RNA (e.g., mRNA)polynucleotide encodes an antigenic polypeptide having at least 95%identity to the amino acid sequence of any one of SEQ ID NO: 10, 14, 18,22, 26, 30, 34, 38, 42 and 45-55. In some embodiments, at least one RNApolynucleotide encodes an antigenic polypeptide having at least 96%identity to the amino acid sequence of any one of SEQ ID NO: 10, 14, 18,22, 26, 30, 34, 38, 42 and 45-55. In some embodiments, at least one RNA(e.g., mRNA) polynucleotide encodes an antigenic polypeptide having atleast 97% identity to the amino acid sequence of any one of SEQ ID NO:10, 14, 18, 22, 26, 30, 34, 38, 42 and 45-55. In some embodiments, atleast one RNA (e.g., mRNA) polynucleotide encodes an antigenicpolypeptide having at least 98% identity to the amino acid sequence ofany one of SEQ ID NO: 10, 14, 18, 22, 26, 30, 34, 38, 42 and 45-55. Insome embodiments, at least one RNA (e.g., mRNA) polynucleotide encodesan antigenic polypeptide having at least 99% identity to the amino acidsequence of any one of SEQ ID NO: 10, 14, 18, 22, 26, 30, 34, 38, 42 and45-55.

In some embodiments, the open reading frame from which the VZVpolypeptide is encoded is codon-optimized. In some embodiments, the atleast one RNA polynucleotide encodes an antigenic protein of any one ofSEQ ID NO: 10, 14, 18, 22, 26, 30, 34, 38, 42 and 45-55, and wherein theRNA polynucleotide is codon-optimized mRNA. In some embodiments, the atleast one RNA polynucleotide comprises a mRNA sequence identified by anyone of SEQ ID NO: 92-108. In some embodiments, the mRNA sequenceidentified by any one of SEQ ID NO: 92-108 is codon optimized to encodeantigenic VZV polypeptides that are as immunogenic, or more immunogenicthan, the antigenic VZV polypeptides encoded by any one of SEQ ID NO:92-108.

In some embodiments, the at least one RNA (e.g., mRNA) polynucleotideencodes an antigenic protein of SEQ ID NO: 10, wherein the RNA (e.g.,mRNA) polynucleotide has less than 80% identity to wild-type mRNAsequence. In some embodiments, the at least one RNA polynucleotideencodes an antigenic protein of SEQ ID NO: 10, wherein the RNA (e.g.,mRNA) polynucleotide has greater than 80% identity to wild-type mRNAsequence, but does not include wild-type mRNA sequence. In someembodiments, the at least one RNA (e.g., mRNA) polynucleotide encodes anantigenic protein of SEQ ID NO: 42, wherein the RNA e.g., mRNA)polynucleotide has less than 80% identity to wild-type mRNA sequence. Insome embodiments, the at least one RNA (e.g., mRNA) polynucleotideencodes an antigenic protein of SEQ ID NO: 42, wherein the RNApolynucleotide has greater than 80% identity to wild-type mRNA sequence,but does not include wild-type mRNA sequence. In some embodiments, theat least one RNA (e.g., mRNA) polynucleotide encodes an antigenicprotein of SEQ ID NO: 14, wherein the RNA (e.g., mRNA) polynucleotidehas less than 80% identity to wild-type mRNA sequence. In someembodiments, the at least one RNA (e.g., mRNA) polynucleotide encodes anantigenic protein of SEQ ID NO: 14, wherein the RNA (e.g., mRNA)polynucleotide has greater than 80% identity to wild-type mRNA sequence,but does not include wild-type mRNA sequence. In some embodiments, theat least one RNA (e.g., mRNA) polynucleotide encodes an antigenicprotein of SEQ ID NO: 26, wherein the RNA polynucleotide has less than80% identity to wild-type mRNA sequence. In some embodiments, the atleast one RNA (e.g., mRNA) polynucleotide encodes an antigenic proteinof SEQ ID NO: 26, wherein the RNA (e.g., mRNA) polynucleotide hasgreater than 80% identity to wild-type mRNA sequence, but does notinclude wild-type mRNA sequence.

In some embodiments, the at least one RNA (e.g., mRNA) polynucleotideencodes an antigenic protein of SEQ ID NO: 30, wherein the RNApolynucleotide has less than 80% identity to wild-type mRNA sequence. Insome embodiments, the at least one RNA polynucleotide encodes anantigenic protein of SEQ ID NO: 30, wherein the RNA (e.g., mRNA)polynucleotide has greater than 80% identity to wild-type mRNA sequence,but does not include wild-type mRNA sequence. In some embodiments, theat least one RNA (e.g., mRNA) polynucleotide is encoded by a sequenceselected from any one of SEQ ID NO: 1-8 and SEQ ID NO 41 and includes atleast one chemical modification.

In some embodiments, the VZV vaccine is multivalent. In someembodiments, the RNA polynucleotide comprises a polynucleotide sequencederived from VZV E1 strain, including, for example, any one or more ofgenotypes E1_32_5, E1_Kel, E1_Dumas, E1_Russia 1999, E1_SD, E1_MSP,E1_36, E1_49, E1_BC, and E1_NH29. In some embodiments, the RNA (e.g.,mRNA) polynucleotide comprises a polynucleotide sequence derived fromVZV E2 strain, including, for example, any one or more of genotypesE2_03-500, E2_2, E2_11, and E2_HJO. In some embodiments, the RNA (e.g.,mRNA) polynucleotide comprises a polynucleotide sequence derived fromVZV J strain, including, for example, genotype pOka. In someembodiments, the RNA (e.g., mRNA) polynucleotide comprises apolynucleotide sequence derived from VZV M1 strain, including, forexample, genotype M1_CA123. In some embodiments, the RNA (e.g., mRNA)polynucleotide comprises a polynucleotide sequence derived from VZV M2strain, including, for example, genotypes M2_8 and M2_DR. In someembodiments, the RNA (e.g., mRNA) polynucleotide comprises apolynucleotide sequence derived from VZV M4 strain, including, forexample, any one or more of genotypes Spain 4242, France 4415, and Italy4053.

Some embodiments of the present disclosure provide a VZV vaccine thatincludes at least one ribonucleic acid (RNA) polynucleotide having anopen reading frame encoding at least one VZV antigenic polypeptide andat least one 5′ terminal cap. In some embodiments, a 5′ terminal cap is7mG(5′)ppp(5′)NlmpNp. Some embodiments of the present disclosure providea VZV vaccine that includes at least one RNA (e.g., mRNA) polynucleotidehaving an open reading frame encoding at least one VZV antigenicpolypeptide, wherein the at least one RNA (e.g., mRNA) polynucleotidehas at least one chemical modification. In some embodiments, the atleast one RNA (e.g., mRNA) polynucleotide further comprises a secondchemical modification. In some embodiments, the at least one RNA (e.g.,mRNA) polynucleotide having at least one chemical modification has a 5′terminal cap. In some embodiments, the at least one chemicalmodification is selected from pseudouridine, N1-methylpseudouridine,N1-ethylpseudouridine, 2-thiouridine, 4′-thiouridine, 5-methylcytosine,2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine,2-thio-5-aza-uridine, 2-thio-dihydropseudouridine,2-thio-dihydrouridine, 2-thio-pseudouridine,4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine,4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine,dihydropseudouridine, 5-methoxyuridine and 2′-O-methyl uridine. In someembodiments, every (100%) of the uridines of the at least one RNApolynucleotide comprises a chemical modification, such as aN1-methylpseudouridine modification or a N1-ethylpseudouridinemodification.

Some embodiments of the present disclosure provide a VZV vaccine thatincludes at least one ribonucleic acid (RNA) polynucleotide having anopen reading frame encoding at least one VZV antigenic polypeptide,wherein at least 80% (e.g., 85%, 90%, 95%, 98%, 99%, 100%) of the uracilin the open reading frame have a chemical modification, optionallywherein the vaccine is formulated in a lipid nanoparticle. In someembodiments, 100% of the uracil in the open reading frame have achemical modification. In some embodiments, a chemical modification isin the 5-position of the uracil. In some embodiments, a chemicalmodification is a N1-methyl pseudouridine. In some embodiments, 100% ofthe uracil in the open reading frame are modified to include N1-methylpseudouridine.

Some embodiments of the present disclosure provide a VZV vaccine that isformulated within a cationic lipid nanoparticle. In some embodiments,the cationic lipid nanoparticle comprises a cationic lipid, aPEG-modified lipid, a sterol and a non-cationic lipid. In someembodiments, a cationic lipid is an ionizable cationic lipid and thenon-cationic lipid is a neutral lipid, and the sterol is a cholesterol.In some embodiments, a cationic lipid is selected from the groupconsisting of 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane(DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate(DLin-MC3-DMA), di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate,(12Z,15Z)—N,N-dimethyl-2-nonylhenicosa-12,15-dien-1-amine, andN,N-dimethyl-1-[(1 S,2R)-2-octylcyclopropyl]heptadecan-8-amine.

In some embodiments, the lipid is

In some embodiments, the lipid is

In some embodiments, at least one cationic lipid selected from compoundsof Formula (I):

or a salt or isomer thereof, wherein:R₁ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀ alkenyl,—R*YR″, —YR″, and —R″M′ R′;R₂ and R₃ are independently selected from the group consisting of H,C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃,together with the atom to which they are attached, form a heterocycle orcarbocycle;R₄ is selected from the group consisting of a C₃₋₆ carbocycle,—(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, —CQ(R)₂, and unsubstituted C₁₋₆alkyl, where Q is selected from a carbocycle, heterocycle, —OR,—O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H, —CXH₂, —CN, —N(R)₂,—C(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂,—N(R)R₈, —O(CH₂)_(n)OR, —N(R)C(═NR₉)N(R)₂, —N(R)C(═CHR₉)N(R)₂,—OC(O)N(R)₂, —N(R)C(O)OR, —N(OR)C(O)R, —N(OR)S(O)₂R, —N(OR)C(O)OR,—N(OR)C(O)N(R)₂, —N(OR)C(S)N(R)₂, —N(OR)C(═NR₉)N(R)₂,—N(OR)C(═CHR₉)N(R)₂, —C(═NR₉)N(R)₂, —C(═NR₉)R, —C(O)N(R)OR, and—C(R)N(R)₂C(O)OR, and each n is independently selected from 1, 2, 3, 4,and 5;each R₅ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;each R₆ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, —S—S—, an aryl group, and a heteroaryl group;R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;R₈ is selected from the group consisting of C₃₋₆ carbocycle andheterocycle;R₉ is selected from the group consisting of H, CN, NO₂, C₁₋₆ alkyl, —OR,—S(O)₂R, —S(O)₂N(R)₂, C₂₋₆ alkenyl, C₃₋₆ carbocycle and heterocycle;each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;each R′ is independently selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl;each R* is independently selected from the group consisting of C₁₋₂alkyl and C₂₋₁₂ alkenyl; each Y is independently a C₃₋₆ carbocycle;each X is independently selected from the group consisting of F, Cl, Br,and I; andm is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13.

In some embodiments, a subset of compounds of Formula (I) includes thosein which when R₄ is —(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, or —CQ(R)₂, then(i) Q is not —N(R)₂ when n is 1, 2, 3, 4 or 5, or (ii) Q is not 5, 6, or7-membered heterocycloalkyl when n is 1 or 2.

In some embodiments, a subset of compounds of Formula (I) includes thosein which R₁ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀alkenyl, —R*YR″, —YR″, and —R″M′R′;

R₂ and R₃ are independently selected from the group consisting of H,C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃,together with the atom to which they are attached, form a heterocycle orcarbocycle;R₄ is selected from the group consisting of a C₃₋₆ carbocycle,—(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, —CQ(R)₂, and unsubstituted C₁₋₆alkyl, where Q is selected from a C₃₋₆ carbocycle, a 5- to 14-memberedheteroaryl having one or more heteroatoms selected from N, O, and S,—OR, —O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H, —CXH₂, —CN,—C(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂,—CRN(R)₂C(O)OR, —N(R)R₈, —O(CH₂)_(n)OR, —N(R)C(═NR₉)N(R)₂,—N(R)C(═CHR₉)N(R)₂, —OC(O)N(R)₂, —N(R)C(O)OR, —N(OR)C(O)R, —N(OR)S(O)₂R,—N(OR)C(O)OR, —N(OR)C(O)N(R)₂, —N(OR)C(S)N(R)₂, —N(OR)C(═NR₉)N(R)₂,—N(OR)C(═CHR₉)N(R)₂, —C(═NR₉)N(R)₂, —C(═NR₉)R, —C(O)N(R)OR, and a 5- to14-membered heterocycloalkyl having one or more heteroatoms selectedfrom N, O, and S which is substituted with one or more substituentsselected from oxo (═O), OH, amino, mono- or di-alkylamino, and C₁₋₃alkyl, and each n is independently selected from 1, 2, 3, 4, and 5;each R₅ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;each R₆ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, —S—S—, an aryl group, and a heteroaryl group;R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;R₈ is selected from the group consisting of C₃₋₆ carbocycle andheterocycle;R₉ is selected from the group consisting of H, CN, NO₂, C₁₋₆ alkyl, —OR,—S(O)₂R, —S(O)₂N(R)₂, C₂₋₆ alkenyl, C₃₋₆ carbocycle and heterocycle;each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H; each R′ is independently selected from thegroup consisting of C₁₋₁₈ alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl;each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₂₋₁₂ alkenyl;each Y is independently a C₃₋₆ carbocycle;each X is independently selected from the group consisting of F, Cl, Br,and I; andm is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,or salts or isomers thereof.

In some embodiments, a subset of compounds of Formula (I) includes thosein which R₁ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀alkenyl, —R*YR″, —YR″, and —R″M′R′;

R₂ and R₃ are independently selected from the group consisting of H,C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃,together with the atom to which they are attached, form a heterocycle orcarbocycle;R₄ is selected from the group consisting of a C₃₋₆ carbocycle,—(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, —CQ(R)₂, and unsubstituted C₁₋₆alkyl, where Q is selected from a C₃₋₆ carbocycle, a 5- to 14-memberedheterocycle having one or more heteroatoms selected from N, O, and S,—OR, —O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H, —CXH₂, —CN,—C(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂,—CRN(R)₂C(O)OR, —N(R)R₈, —O(CH₂)_(n)OR, —N(R)C(═NR₉)N(R)₂,—N(R)C(═CHR₉)N(R)₂, —OC(O)N(R)₂, —N(R)C(O)OR, —N(OR)C(O)R, —N(OR)S(O)₂R,—N(OR)C(O)OR, —N(OR)C(O)N(R)₂, —N(OR)C(S)N(R)₂, —N(OR)C(═NR₉)N(R)₂,—N(OR)C(═CHR₉)N(R)₂, —C(═NR₉)R, —C(O)N(R)OR, and —C(═NR₉)N(R)₂, and eachn is independently selected from 1, 2, 3, 4, and 5; and when Q is a 5-to 14-membered heterocycle and (i) R₄ is —(CH₂)_(n)Q in which n is 1 or2, or (ii) R₄ is —(CH₂)_(n)CHQR in which n is 1, or (iii) R₄ is —CHQR,and —CQ(R)₂, then Q is either a 5- to 14-membered heteroaryl or 8- to14-membered heterocycloalkyl;each R₅ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;each R₆ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, —S—S—, an aryl group, and a heteroaryl group;R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;R₈ is selected from the group consisting of C₃₋₆ carbocycle andheterocycle;R₉ is selected from the group consisting of H, CN, NO₂, C₁₋₆ alkyl, —OR,—S(O)₂R, —S(O)₂N(R)₂, C₂₋₆ alkenyl, C₃₋₆ carbocycle and heterocycle;each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;each R′ is independently selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl;each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₂₋₁₂ alkenyl;each Y is independently a C₃₋₆ carbocycle;each X is independently selected from the group consisting of F, Cl, Br,and I; andm is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,or salts or isomers thereof.

In some embodiments, a subset of compounds of Formula (I) includes thosein which

R₁ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀ alkenyl,—R*YR″, —YR″, and —R″M′R′;R₂ and R₃ are independently selected from the group consisting of H,C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃,together with the atom to which they are attached, form a heterocycle orcarbocycle;R₄ is selected from the group consisting of a C₃₋₆ carbocycle,—(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, —CQ(R)₂, and unsubstituted C₁₋₆alkyl, where Q is selected from a C₃₋₆ carbocycle, a 5- to 14-memberedheteroaryl having one or more heteroatoms selected from N, O, and S,—OR, —O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H, —CXH₂, —CN,—C(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂,—CRN(R)₂C(O)OR, —N(R)R₈, —O(CH₂)_(n)OR, —N(R)C(═NR₉)N(R)₂,—N(R)C(═CHR₉)N(R)₂, —OC(O)N(R)₂, —N(R)C(O)OR, —N(OR)C(O)R, —N(OR)S(O)₂R,—N(OR)C(O)OR, —N(OR)C(O)N(R)₂, —N(OR)C(S)N(R)₂, —N(OR)C(═NR₉)N(R)₂,—N(OR)C(═CHR₉)N(R)₂, —C(═NR₉)R, —C(O)N(R)OR, and —C(═NR₉)N(R)₂, and eachn is independently selected from 1, 2, 3, 4, and 5;each R₅ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;each R₆ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, —S—S—, an aryl group, and a heteroaryl group;R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;R₈ is selected from the group consisting of C₃₋₆ carbocycle andheterocycle;R₉ is selected from the group consisting of H, CN, NO₂, C₁₋₆ alkyl, —OR,—S(O)₂R, —S(O)₂N(R)₂, C₂₋₆ alkenyl, C₃₋₆ carbocycle and heterocycle;each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;each R′ is independently selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl;each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₂₋₁₂ alkenyl;each Y is independently a C₃₋₆ carbocycle;each X is independently selected from the group consisting of F, Cl, Br,and I; andm is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,or salts or isomers thereof.

In some embodiments, a subset of compounds of Formula (I) includes thosein which

R₁ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀ alkenyl,—R*YR″, —YR″, and —R″M′R′;R₂ and R₃ are independently selected from the group consisting of H,C₂₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃,together with the atom to which they are attached, form a heterocycle orcarbocycle;R₄ is —(CH₂)_(n)Q or —(CH₂)_(n)CHQR, where Q is —N(R)₂, and n isselected from 3, 4, and 5;each R₅ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;each R₆ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, —S—S—, an aryl group, and a heteroaryl group; R₇ is selectedfrom the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl, and H;each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;each R′ is independently selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl;each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₁₋₁₂ alkenyl;each Y is independently a C₃₋₆ carbocycle;each X is independently selected from the group consisting of F, Cl, Br,and I; andm is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,or salts or isomers thereof.

In some embodiments, a subset of compounds of Formula (I) includes thosein which

R₁ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀ alkenyl,—R*YR″, —YR″, and —R″M′R′;R₂ and R₃ are independently selected from the group consisting of C₁₋₁₄alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃, togetherwith the atom to which they are attached, form a heterocycle orcarbocycle;R₄ is selected from the group consisting of —(CH₂)_(n)Q, —(CH₂)_(n)CHQR,—CHQR, and —CQ(R)₂, where Q is —N(R)₂, and n is selected from 1, 2, 3,4, and 5;each R₅ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;each R₆ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, —S—S—, an aryl group, and a heteroaryl group;R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;each R′ is independently selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl;each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₁₋₁₂ alkenyl;each Y is independently a C₃₋₆ carbocycle;each X is independently selected from the group consisting of F, Cl, Br,and I; andm is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,or salts or isomers thereof.

In some embodiments, a subset of compounds of Formula (I) includes thoseof Formula (IA):

or a salt or isomer thereof, wherein l is selected from 1, 2, 3, 4, and5; m is selected from 5, 6, 7, 8, and 9; M₁ is a bond or M′; R₄ isunsubstituted C₁₋₃ alkyl, or —(CH₂)_(n)Q, in which Q is OH,—NHC(S)N(R)₂, —NHC(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)Rs,—NHC(═NR₉)N(R)₂, —NHC(═CHR₉)N(R)₂, —OC(O)N(R)₂, —N(R)C(O)OR, heteroarylor heterocycloalkyl; M and M′ are independently selected from —C(O)O—,—OC(O)—, —C(O)N(R′)—, —P(O)(OR′)O—, —S—S—, an aryl group, and aheteroaryl group; and R₂ and R₃ are independently selected from thegroup consisting of H, C₁₋₁₄ alkyl, and C₂₋₁₄ alkenyl.

In some embodiments, the cationic lipid nanoparticle has a molar ratioof about 20-60% cationic lipid, about 5-25% non-cationic lipid, about25-55% sterol, and about 0.5-15% PEG-modified lipid. In someembodiments, the nanoparticle has a polydispersity value of less than0.4. In some embodiments, the nanoparticle has a net neutral charge at aneutral pH value. In some embodiments, the nanoparticle has a meandiameter of 50-200 nm.

Some embodiments of the present disclosure provide methods of inducingan antigen specific immune response in a subject, comprisingadministering to the subject a VZV RNA (e.g., mRNA) vaccine in an amounteffective to produce an antigen specific immune response. In someembodiments, an antigen specific immune response comprises a T cellresponse or a B cell response. In some embodiments, an antigen specificimmune response comprises a T cell response and a B cell response. Insome embodiments, a method of producing an antigen specific immuneresponse involves a single administration of the vaccine. In someembodiments, a method further includes administering to the subject abooster dose of the vaccine. In some embodiments, a vaccine isadministered to the subject by intradermal or intramuscular injection.

Also provided herein are VZV RNA (e.g., mRNA) vaccines for use in amethod of inducing an antigen specific immune response in a subject, themethod comprising administering the vaccine to the subject in an amounteffective to produce an antigen specific immune response.

Further provided herein are uses of VZV RNA (e.g., mRNA) vaccines in themanufacture of a medicament for use in a method of inducing an antigenspecific immune response in a subject, the method comprisingadministering the vaccine to the subject in an amount effective toproduce an antigen specific immune response.

Some aspects of the present disclosure provide methods of preventing ortreating VZV infection comprising administering to a subject the VZV RNA(e.g., mRNA) vaccine of the present disclosure. In some embodiments, theVZV RNA (e.g., mRNA) vaccine is formulated in an effective amount toproduce an antigen specific immune response in a subject.

Some embodiments of the present disclosure provide methods of inducingan antigen specific immune response in a subject, the methods comprisingadministering to a subject a VZV RNA (e.g., mRNA) vaccine as providedherein in an effective amount to produce an antigen specific immuneresponse in a subject.

In some embodiments, an anti-VZV antigenic polypeptide antibody titerproduced in the subject is increased by at least 1 log relative to acontrol. In some embodiments, the anti-VZV antigenic polypeptideantibody titer produced in the subject is increased by 1-3 log relativeto a control.

In some embodiments, the anti-VZV antigenic polypeptide antibody titerproduced in the subject is increased at least 2 times relative to acontrol. In some embodiments, the anti-VZV antigenic polypeptideantibody titer produced in the subject is increased at least 5 timesrelative to a control. In some embodiments, the anti-VZV antigenicpolypeptide antibody titer produced in the subject is increased at least10 times relative to a control. In some embodiments, the anti-VZVantigenic polypeptide antibody titer produced in the subject isincreased 2-10 times relative to a control.

In some embodiments, the control is an anti-VZV antigenic polypeptideantibody titer produced in a subject who has not been administered VZVvaccine. In some embodiments, the control is an anti-VZV antigenicpolypeptide antibody titer produced in a subject who has beenadministered a live attenuated or inactivated VZV vaccine. In someembodiments, the control is an anti-VZV antigenic polypeptide antibodytiter produced in a subject who has been administered a recombinant orpurified VZV protein vaccine. In some embodiments, the control is ananti-VZV antigenic polypeptide antibody titer produced in a subject whohas been administered an VZV virus-like particle (VLP) vaccine.

In some embodiments, the effective amount is a dose equivalent to atleast a 2-fold reduction in the standard of care dose of a recombinantVZV protein vaccine, wherein an anti-VZV antigenic polypeptide antibodytiter produced in the subject is equivalent to an anti-VZV antigenicpolypeptide antibody titer produced in a control subject administeredthe standard of care dose of a recombinant or purified VZV proteinvaccine, a live attenuated or inactivated VZV vaccine, or a VZV VLPvaccine.

In some embodiments, the effective amount is a dose equivalent to atleast a 4-fold reduction in the standard of care dose of a recombinantVZV protein vaccine, wherein an anti-VZV antigenic polypeptide antibodytiter produced in the subject is equivalent to an anti-VZV antigenicpolypeptide antibody titer produced in a control subject administeredthe standard of care dose of a recombinant or purified VZV proteinvaccine, a live attenuated or inactivated VZV vaccine, or a VZV VLPvaccine.

In some embodiments, the effective amount is a dose equivalent to atleast a 10-fold reduction in the standard of care dose of a recombinantVZV protein vaccine, wherein an anti-VZV antigenic polypeptide antibodytiter produced in the subject is equivalent to an anti-VZV antigenicpolypeptide antibody titer produced in a control subject administeredthe standard of care dose of a recombinant or purified VZV proteinvaccine, a live attenuated or inactivated VZV vaccine, or a VZV VLPvaccine.

In some embodiments, the effective amount is a dose equivalent to atleast a 100-fold reduction in the standard of care dose of a recombinantVZV protein vaccine, wherein an anti-VZV antigenic polypeptide antibodytiter produced in the subject is equivalent to an anti-VZV antigenicpolypeptide antibody titer produced in a control subject administeredthe standard of care dose of a recombinant or purified VZV proteinvaccine, a live attenuated or inactivated VZV vaccine, or a VZV VLPvaccine.

In some embodiments, the effective amount is a dose equivalent to atleast a 1000-fold reduction in the standard of care dose of arecombinant VZV protein vaccine, wherein an anti-VZV antigenicpolypeptide antibody titer produced in the subject is equivalent to ananti-VZV antigenic polypeptide antibody titer produced in a controlsubject administered the standard of care dose of a recombinant orpurified VZV protein vaccine, a live attenuated or inactivated VZVvaccine, or a VZV VLP vaccine.

In some embodiments, the effective amount is a dose equivalent to a2-fold to 1000-fold reduction in the standard of care dose of arecombinant VZV protein vaccine, wherein an anti-VZV antigenicpolypeptide antibody titer produced in the subject is equivalent to ananti-VZV antigenic polypeptide antibody titer produced in a controlsubject administered the standard of care dose of a recombinant orpurified VZV protein vaccine, a live attenuated or inactivated VZVvaccine, or a VZV VLP vaccine.

In some embodiments, the effective amount is a total dose of 25 μg to1000 μg, or 50 g to 1000 μg or 25 to 200 μg. In some embodiments, theeffective amount is a total dose of 50 μg, 100 μg, 200 μg, 400 μg, 800μg, or 1000 μg. In some embodiments, the effective amount is a totaldose of 200 μg. In some embodiments, the effective amount is a totaldose of 50 μg to 400 μg. In some embodiments, the effective amount is atotal dose of 50 μg, 100 Gg, 150 μg, 200 μg, 250 μg, 300 μg, 350 μg or400 μg. In some embodiments, the effective amount is a dose of 25 μgadministered to the subject a total of two times. In some embodiments,the effective amount is a dose of 50 μg administered to the subject atotal of two times. In some embodiments, the effective amount is a doseof 100 μg administered to the subject a total of two times. In someembodiments, the effective amount is a dose of 200 g administered to thesubject a total of two times. In some embodiments, the effective amountis a dose of 400 μg administered to the subject a total of two times. Insome embodiments, the effective amount is a dose of 500 μg administeredto the subject a total of two times.

In some embodiments, the efficacy (or effectiveness) of the VZV RNA(e.g., mRNA) vaccine against VZV is greater than 60%.

Vaccine efficacy may be assessed using standard analyses (see, e.g.,Weinberg et al., J Infect Dis. 2010 June 1; 201(11): 1607-10). Forexample, vaccine efficacy may be measured by double-blind, randomized,clinical controlled trials. Vaccine efficacy may be expressed as aproportionate reduction in disease attack rate (AR) between theunvaccinated (ARU) and vaccinated (ARV) study cohorts and can becalculated from the relative risk (RR) of disease among the vaccinatedgroup with use of the following formulas:

Efficacy=(ARU−ARV)/ARU×100; and

Efficacy=(1−RR)×100.

Likewise, vaccine effectiveness may be assessed using standard analyses(see, e.g., Weinberg et al., J Infect Dis. 2010 Jun. 1;201(11):1607-10). Vaccine effectiveness is an assessment of how avaccine (which may have already proven to have high vaccine efficacy)reduces disease in a population. This measure can assess the net balanceof benefits and adverse effects of a vaccination program, not just thevaccine itself, under natural field conditions rather than in acontrolled clinical trial. Vaccine effectiveness is proportional tovaccine efficacy (potency) but is also affected by how well targetgroups in the population are immunized, as well as by othernon-vaccine-related factors that influence the ‘real-world’ outcomes ofhospitalizations, ambulatory visits, or costs. For example, aretrospective case control analysis may be used, in which the rates ofvaccination among a set of infected cases and appropriate controls arecompared. Vaccine effectiveness may be expressed as a rate difference,with use of the odds ratio (OR) for developing infection despitevaccination:

Effectiveness=(1−OR)×100.

In some embodiments, the efficacy (or effectiveness) of the VZV RNA(e.g., mRNA) vaccine against VZV is greater than 65%. In someembodiments, the efficacy (or effectiveness) of the vaccine against VZVis greater than 70%. In some embodiments, the efficacy (oreffectiveness) of the vaccine against VZV is greater than 75%. In someembodiments, the efficacy (or effectiveness) of the vaccine against VZVis greater than 80%. In some embodiments, the efficacy (oreffectiveness) of the vaccine against VZV is greater than 85%. In someembodiments, the efficacy (or effectiveness) of the vaccine against VZVis greater than 90%.

In some embodiments, the vaccine immunizes the subject against VZV up to1 year (e.g. for a single VZV season). In some embodiments, the vaccineimmunizes the subject against VZV for up to 2 years. In someembodiments, the vaccine immunizes the subject against VZV for more than2 years. In some embodiments, the vaccine immunizes the subject againstVZV for more than 3 years. In some embodiments, the vaccine immunizesthe subject against VZV for more than 4 years. In some embodiments, thevaccine immunizes the subject against VZV for 5-10 years.

In some embodiments, the subject administered an VZV RNA (e.g., mRNA)vaccine is between the ages of about 12 months old and about 10 yearsold (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 years old). In someembodiments, the subject administered an VZV RNA (e.g., mRNA) vaccine isbetween the ages of about 12 months old and about 15 months old (e.g.,about 12, 12.5, 13, 13.5, 14, 14.5 or 15 months old). In someembodiments, the subject administered an VZV RNA (e.g., mRNA) vaccine isbetween the ages of about 4 years old and about 6 years old (e.g., about4, 4.5, 5, 5.6, or 6 years old).

In some embodiments, the subject is a young adult between the ages ofabout 20 years and about 50 years (e.g., about 20, 25, 30, 35, 40, 45 or50 years old).

In some embodiments, the subject is an elderly subject about 60 yearsold, about 70 years old, or older (e.g., about 60, 65, 70, 75, 80, 85 or90 years old).

In some embodiments, the subject has been exposed to VZV, is infectedwith (has) VZV, or is at risk of infection by VZV.

In some embodiments, the subject is immunocompromised (has an impairedimmune system, e.g., has an immune disorder or autoimmune disorder).

Some aspects of the present disclosure provide varicella zoster virus(VZV) RNA (e.g., mRNA) vaccines containing a signal peptide linked to aVZV antigenic polypeptide.

Thus, in some embodiments, the VZV RNA (e.g., mRNA) vaccines contain atleast one ribonucleic acid (RNA) polynucleotide having an open readingframe encoding a signal peptide linked to a VZV antigenic peptide. Alsoprovided herein are nucleic acids encoding the VZV RNA (e.g., mRNA)vaccines disclosed herein.

Other aspects of the present disclosure provide varicella zoster virus(VZV) vaccines containing a signal peptide linked to a VZV antigenicpolypeptide. In some embodiments, the VZV antigenic polypeptide is a VZVglycoprotein. In some embodiments, the VZV glycoprotein is selected fromVZV gE, gI, gB, gH, gK, gL, gC, gN, and gM. In some embodiments, the VZVglycoprotein is VZV gE or a variant VZV gE polypeptide.

In some embodiments, the signal peptide is a IgE signal peptide. In someembodiments, the signal peptide is an IgE HC (Ig heavy chain epsilon-1)signal peptide. In some embodiments, the signal peptide has the sequenceMDWTWILFLVAAATRVHS (SEQ ID NO: 56). In some embodiments, the signalpeptide is an IgGK signal peptide. In some embodiments, the signalpeptide has the sequence METPAQLLFLLLLWLPDTTG (SEQ ID NO: 57). In someembodiments, the signal peptide is selected from: a Japaneseencephalitis PRM signal sequence (MLGSNSGQRVVFTILLLLVAPAYS; SEQ ID NO:109), VSVg protein signal sequence (MKCLLYLAFLFIGVNCA; SEQ ID NO: 110)and Japanese encephalitis JEV signal sequence (MWLVSLAIVTACAGA; SEQ IDNO: 111).

Further provided herein are nucleic acids encoding VZV vaccinesdisclosed herein. Such VZV vaccines include at least one ribonucleicacid (RNA) (e.g., mRNA) polynucleotide having an open reading frameencoding a signal peptide linked to a VZV antigenic polypeptide. In someembodiments, the VZV antigenic peptide is a VZV glycoprotein. In someembodiments, the VZV glycoprotein is selected from VZV gE, gI, gB, gH,gK, gL, gC, gN, and gM. In some embodiments, the VZV antigenic peptideis a VZV gE or a variant of the gE polypeptide.

In some embodiments, an effective amount of an VZV RNA (e.g., mRNA)vaccine (e.g., a single dose of the VZV vaccine) results in a 2 fold to200 fold (e.g., about 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60,70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200fold) increase in serum neutralizing antibodies against VZV, relative toa control. In some embodiments, a single dose of the VZV RNA (e.g.,mRNA) vaccine results in an about 5 fold, 50 fold, or 150 fold increasein serum neutralizing antibodies against VZV, relative to a control. Insome embodiments, a single dose of the VZV RNA (e.g., mRNA) vaccineresults in an about 2 fold to 10 fold, or an about 40 to 60 foldincrease in serum neutralizing antibodies against VZV, relative to acontrol.

In some embodiments, efficacy of RNA vaccines RNA (e.g., mRNA) can besignificantly enhanced when combined with a flagellin adjuvant, inparticular, when one or more antigen-encoding mRNAs is combined with anmRNA encoding flagellin.

RNA (e.g., mRNA) vaccines combined with the flagellin adjuvant (e.g.,mRNA-encoded flagellin adjuvant) have superior properties in that theymay produce much larger antibody titers and produce responses earlierthan commercially available vaccine formulations. While not wishing tobe bound by theory, it is believed that the RNA vaccines, for example,as mRNA polynucleotides, are better designed to produce the appropriateprotein conformation upon translation, for both the antigen and theadjuvant, as the RNA (e.g., mRNA) vaccines co-opt natural cellularmachinery. Unlike traditional vaccines, which are manufactured ex vivoand may trigger unwanted cellular responses, RNA (e.g., mRNA) vaccinesare presented to the cellular system in a more native fashion.

Some embodiments of the present disclosure provide RNA (e.g., mRNA)vaccines that include at least one RNA (e.g., mRNA) polynucleotidehaving an open reading frame encoding at least one antigenic polypeptideand at least one RNA (e.g., mRNA polynucleotide) having an open readingframe encoding a flagellin adjuvant.

In some embodiments, at least one flagellin polypeptide (e.g., encodedflagellin polypeptide) is a flagellin protein. In some embodiments, atleast one flagellin polypeptide (e.g., encoded flagellin polypeptide) isan immunogenic flagellin fragment. In some embodiments, at least oneflagellin polypeptide and at least one antigenic polypeptide are encodedby a single RNA (e.g., mRNA) polynucleotide. In other embodiments, atleast one flagellin polypeptide and at least one antigenic polypeptideare each encoded by a different RNA polynucleotide.

In some embodiments at least one flagellin polypeptide has at least 80%,at least 85%, at least 90%, or at least 95% identity to a flagellinpolypeptide having a sequence of any one of SEQ ID NO: 115-117.

In some embodiments the nucleic acid vaccines described herein arechemically modified. In other embodiments the nucleic acid vaccines areunmodified.

In some embodiments, the RNA polynucleotide is any one of SEQ ID NO: 1-8and 41 and includes at least one chemical modification. In otherembodiments, the RNA polynucleotide is any one of SEQ ID NO: 1-8 and 41and does not include any nucleotide modifications, or is unmodified. Inyet other embodiments, the at least one RNA polynucleotide encodes anantigenic protein of any one of SEQ ID NO: 10, 14, 18, 22, 26, 30, 34,38, 42 and 45-55 and includes at least one chemical modification. Inother embodiments, the RNA polynucleotide encodes an antigenic proteinof any one of SEQ ID NO: 10, 14, 18, 22, 26, 30, 34, 38, 42 and 45-55and does not include any nucleotide modifications, or is unmodified.

In some embodiments, the RNA polynucleotide comprises a sequence of anyone of SEQ ID NO: 142-150. In some embodiments, the RNA polynucleotidecomprises a sequence of any one of SEQ ID NO: 142-150 and includes atleast one chemical modification. In other embodiments, the RNApolynucleotide comprises a sequence of any one of SEQ ID NO: 142-150 anddoes not include any nucleotide modifications, or is unmodified.

Yet other aspects provide compositions for and methods of vaccinating asubject comprising administering to the subject a nucleic acid vaccinecomprising one or more RNA polynucleotides having an open reading frameencoding a first antigenic VZV polypeptide, wherein the RNApolynucleotide does not include a stabilization element, and wherein anadjuvant is not co-formulated or co-administered with the vaccine.

In other aspects the invention is a composition for or method ofvaccinating a subject comprising administering to the subject a nucleicacid vaccine comprising one or more RNA polynucleotides having an openreading frame encoding a first antigenic polypeptide wherein a dosage ofbetween 10 μg/kg and 400 μg/kg of the nucleic acid vaccine isadministered to the subject. In some embodiments the dosage of the RNApolynucleotide is 1-5 μg, 5-10 μg, 10-15 μg, 15-20 μg, 10-25 μg, 20-25μg, 20-50 μg, 30-50 μg, 40-50 μg, 40-60 μg, 60-80 μg, 60-100 μg, 50-100μg, 80-120 μg, 40-120 μg, 40-150 μg, 50-150 μg, 50-200 μg, 80-200 μg,100-200 μg, 120-250 μg, 150-250 μg, 180-280 μg, 200-300 μg, 50-300 μg,80-300 μg, 100-300 μg, 40-300 μg, 50-350 μg, 100-350 μg, 200-350 μg,300-350 μg, 320-400 μg, 40-380 μg, 40-100 μg, 100-400 μg, 200-400 μg, or300-400 μg per dose. In some embodiments, the nucleic acid vaccine isadministered to the subject by intradermal or intramuscular injection.In some embodiments, the nucleic acid vaccine is administered to thesubject on day zero. In some embodiments, a second dose of the nucleicacid vaccine is administered to the subject on day twenty one.

In some embodiments, a dosage of 25 micrograms of the RNA polynucleotideis included in the nucleic acid vaccine administered to the subject. Insome embodiments, a dosage of 100 micrograms of the RNA polynucleotideis included in the nucleic acid vaccine administered to the subject. Insome embodiments, a dosage of 50 micrograms of the RNA polynucleotide isincluded in the nucleic acid vaccine administered to the subject. Insome embodiments, a dosage of 75 micrograms of the RNA polynucleotide isincluded in the nucleic acid vaccine administered to the subject. Insome embodiments, a dosage of 150 micrograms of the RNA polynucleotideis included in the nucleic acid vaccine administered to the subject. Insome embodiments, a dosage of 400 micrograms of the RNA polynucleotideis included in the nucleic acid vaccine administered to the subject. Insome embodiments, a dosage of 200 micrograms of the RNA polynucleotideis included in the nucleic acid vaccine administered to the subject. Insome embodiments, the RNA polynucleotide accumulates at a 100 foldhigher level in the local lymph node in comparison with the distal lymphnode. In other embodiments the nucleic acid vaccine is chemicallymodified and in other embodiments the nucleic acid vaccine is notchemically modified.

Aspects of the invention provide a nucleic acid vaccine comprising oneor more RNA polynucleotides having an open reading frame encoding afirst antigenic polypeptide, wherein the RNA polynucleotide does notinclude a stabilization element, and a pharmaceutically acceptablecarrier or excipient, wherein an adjuvant is not included in thevaccine. In some embodiments, the stabilization element is a histonestem-loop. In some embodiments, the stabilization element is a nucleicacid sequence having increased GC content relative to wild typesequence.

Aspects of the invention provide nucleic acid vaccines comprising one ormore RNA polynucleotides having an open reading frame encoding a firstantigenic polypeptide, wherein the RNA polynucleotide is present in theformulation for in vivo administration to a host, which confers anantibody titer superior to the criterion for seroprotection for thefirst antigen for an acceptable percentage of human subjects. In someembodiments, the antibody titer produced by the mRNA vaccines of theinvention is a neutralizing antibody titer. In some embodiments theneutralizing antibody titer is greater than a protein vaccine. In otherembodiments the neutralizing antibody titer produced by the mRNAvaccines of the invention is greater than an adjuvanted protein vaccine.In yet other embodiments the neutralizing antibody titer produced by themRNA vaccines of the invention is 1,000-10,000, 1,200-10,000,1,400-10,000, 1,500-10,000, 1,000-5,000, 1,000-4,000, 1,800-10,000,2000-10,000, 2,000-5,000, 2,000-3,000, 2,000-4,000, 3,000-5,000,3,000-4,000, or 2,000-2,500. A neutralization titer is typicallyexpressed as the highest serum dilution required to achieve a 50%reduction in the number of plaques.

Also provided are nucleic acid vaccines comprising one or more RNApolynucleotides having an open reading frame encoding a first antigenicpolypeptide, wherein the RNA polynucleotide is present in a formulationfor in vivo administration to a host for eliciting a longer lasting highantibody titer than an antibody titer elicited by an mRNA vaccine havinga stabilizing element or formulated with an adjuvant and encoding thefirst antigenic polypeptide. In some embodiments, the RNA polynucleotideis formulated to produce a neutralizing antibodies within one week of asingle administration. In some embodiments, the adjuvant is selectedfrom a cationic peptide and an immunostimulatory nucleic acid. In someembodiments, the cationic peptide is protamine.

Aspects provide nucleic acid vaccines comprising one or more RNApolynucleotides having an open reading frame comprising at least onechemical modification or optionally no chemical modification, the openreading frame encoding a first antigenic polypeptide, wherein the RNApolynucleotide is present in the formulation for in vivo administrationto a host such that the level of antigen expression in the hostsignificantly exceeds a level of antigen expression produced by an mRNAvaccine having a stabilizing element or formulated with an adjuvant andencoding the first antigenic polypeptide.

Other aspects provide nucleic acid vaccines comprising one or more RNApolynucleotides having an open reading frame comprising at least onechemical modification or optionally no chemical modification, the openreading frame encoding a first antigenic polypeptide, wherein thevaccine has at least 10 fold less RNA polynucleotide than is requiredfor an unmodified mRNA vaccine to produce an equivalent antibody titer.In some embodiments, the RNA polynucleotide is present in a dosage of25-100 micrograms.

Aspects of the invention also provide a unit of use vaccine, comprisingbetween 10 ug and 400 μg of one or more RNA polynucleotides having anopen reading frame comprising at least one chemical modification oroptionally no chemical modification, the open reading frame encoding afirst antigenic polypeptide, and a pharmaceutically acceptable carrieror excipient, formulated for delivery to a human subject. In someembodiments, the vaccine further comprises a cationic lipidnanoparticle.

Aspects of the invention provide methods of creating, maintaining orrestoring antigenic memory to a VZV strain in an individual orpopulation of individuals comprising administering to said individual orpopulation an antigenic memory booster nucleic acid vaccine comprising(a) at least one RNA polynucleotide, said polynucleotide comprising atleast one chemical modification or optionally no chemical modificationand two or more codon-optimized open reading frames, said open readingframes encoding a set of reference antigenic polypeptides, and (b)optionally a pharmaceutically acceptable carrier or excipient. In someembodiments, the vaccine is administered to the individual via a routeselected from the group consisting of intramuscular administration,intradermal administration and subcutaneous administration. In someembodiments, the administering step comprises contacting a muscle tissueof the subject with a device suitable for injection of the composition.In some embodiments, the administering step comprises contacting amuscle tissue of the subject with a device suitable for injection of thecomposition in combination with electroporation.

Aspects of the invention provide methods of vaccinating a subjectcomprising administering to the subject a single dosage of between 25μg/kg and 400 μg/kg of a nucleic acid vaccine comprising one or more RNApolynucleotides having an open reading frame encoding a first antigenicpolypeptide in an effective amount to vaccinate the subject.

Other aspects provide nucleic acid vaccines comprising one or more RNApolynucleotides having an open reading frame comprising at least onechemical modification, the open reading frame encoding a first antigenicpolypeptide, wherein the vaccine has at least 10 fold less RNApolynucleotide than is required for an unmodified mRNA vaccine toproduce an equivalent antibody titer. In some embodiments, the RNApolynucleotide is present in a dosage of 25-100 micrograms.

Other aspects provide nucleic acid vaccines comprising an LNP formulatedRNA polynucleotide having an open reading frame comprising no modifiednucleotides (unmodified), the open reading frame encoding a firstantigenic polypeptide, wherein the vaccine has at least 10 fold less RNApolynucleotide than is required for an unmodified mRNA vaccine notformulated in a LNP to produce an equivalent antibody titer. In someembodiments, the RNA polynucleotide is present in a dosage of 25-100micrograms.

The data presented in the Examples demonstrate significant enhancedimmune responses using the formulations of the invention. The datademonstrated the effectiveness of both chemically modified andunmodified RNA vaccines of the invention. Surprisingly, in contrast toprior art reports that it was preferable to use chemically unmodifiedmRNA formulated in a carrier for the production of vaccines, it wasdiscovered herein that chemically modified mRNA-LNP vaccines required amuch lower effective mRNA dose than unmodified mRNA, i.e., tenfold lessthan unmodified mRNA when formulated in carriers other than LNP. Boththe chemically modified and unmodified RNA vaccines of the inventionproduce better immune responses than mRNA vaccines formulated in adifferent lipid carrier.

In other aspects the invention encompasses a method of treating anelderly subject age 60 years or older comprising administering to thesubject a nucleic acid vaccine comprising one or more RNApolynucleotides having an open reading frame encoding a VZV antigenicpolypeptide in an effective amount to vaccinate the subject.

In other aspects the invention encompasses a method of treating a youngsubject age 17 years or younger comprising administering to the subjecta nucleic acid vaccine comprising one or more RNA polynucleotides havingan open reading frame encoding a VZV antigenic polypeptide in aneffective amount to vaccinate the subject.

In other aspects the invention encompasses a method of treating an adultsubject comprising administering to the subject a nucleic acid vaccinecomprising one or more RNA polynucleotides having an open reading frameencoding a VZV antigenic polypeptide in an effective amount to vaccinatethe subject.

In preferred aspects, vaccines of the invention (e.g., LNP-encapsulatedmRNA vaccines) produce prophylactically- and/ortherapeutically-efficacious levels, concentrations and/or titers ofantigen-specific antibodies in the blood or serum of a vaccinatedsubject. As defined herein, the term antibody titer refers to the amountof antigen-specific antibody produces in s subject, e.g., a humansubject. In exemplary embodiments, antibody titer is expressed as theinverse of the greatest dilution (in a serial dilution) that still givesa positive result. In exemplary embodiments, antibody titer isdetermined or measured by enzyme-linked immunosorbent assay (ELISA). Inexemplary embodiments, antibody titer is determined or measured byneutralization assay, e.g., by microneutralization assay. In certainaspects, antibody titer measurement is expressed as a ratio, such as1:40, 1:100, etc.

In exemplary embodiments of the invention, an efficacious vaccineproduces an antibody titer of greater than 1:40, greater that 1:100,greater than 1:400, greater than 1:1000, greater than 1:2000, greaterthan 1:3000, greater than 1:4000, greater than 1:500, greater than1:6000, greater than 1:7500, greater than 1:10000. In exemplaryembodiments, the antibody titer is produced or reached by 10 daysfollowing vaccination, by 20 days following vaccination, by 30 daysfollowing vaccination, by 40 days following vaccination, or by 50 ormore days following vaccination. In exemplary embodiments, the titer isproduced or reached following a single dose of vaccine administered tothe subject. In other embodiments, the titer is produced or reachedfollowing multiple doses, e.g., following a first and a second dose(e.g., a booster dose.) In exemplary aspects of the invention,antigen-specific antibodies are measured in units of μg/ml or aremeasured in units of IU/L (International Units per liter) or mIU/ml(milli International Units per ml). In exemplary embodiments of theinvention, an efficacious vaccine produces >0.5 μg/ml, >0.1 μg/ml, >0.2μg/ml, >0.35 μg/ml, >0.5 μg/ml, >1 μg/ml, >2 μg/ml, >5 μg/ml or >10μg/ml. In exemplary embodiments of the invention, an efficacious vaccineproduces >10 mIU/ml, >20 mIU/ml, >50 mIU/ml, >100 mIU/ml, >200mIU/ml, >500 mIU/ml or >1000 mIU/ml. In exemplary embodiments, theantibody level or concentration is produced or reached by 10 daysfollowing vaccination, by 20 days following vaccination, by 30 daysfollowing vaccination, by 40 days following vaccination, or by 50 ormore days following vaccination. In exemplary embodiments, the level orconcentration is produced or reached following a single dose of vaccineadministered to the subject. In other embodiments, the level orconcentration is produced or reached following multiple doses, e.g.,following a first and a second dose (e.g., a booster dose.) In exemplaryembodiments, antibody level or concentration is determined or measuredby enzyme-linked immunosorbent assay (ELISA). In exemplary embodiments,antibody level or concentration is determined or measured byneutralization assay, e.g., by microneutralization assay.

Each of the limitations of the invention can encompass variousembodiments of the invention. It is, therefore, anticipated that each ofthe limitations of the invention involving any one element orcombinations of elements can be included in each aspect of theinvention.

This invention is not limited in its application to the details ofconstruction and the arrangement of components set forth in thefollowing description or illustrated in the drawings. The invention iscapable of other embodiments and of being practiced or of being carriedout in various ways.

BRIEF DESCRIPTION OF DRAWINGS

The accompanying drawings are not intended to be drawn to scale. In thedrawings, each identical or nearly identical component that isillustrated in various figures is represented by a like numeral. Forpurposes of clarity, not every component may be labeled in everydrawing. In the drawings:

FIG. 1 is a schematic depicting a proposed Varicella zoster viruspathway.

FIG. 2 is a schematic of the constructs encoding VZV gE (strain Oka).

FIG. 3 depicts the study design and injection schedule for theimmunization of BALB/C mice with MC3 formulated mRNA encoded VZV gEantigens.

FIG. 4 is a schematic showing various variant VZV gE antigens. Thisfigure depicts SEQ ID NOs: 120, 132, and 58 from top to bottom,respectively.

FIG. 5 is a graph showing the results of an ELISA assay that shows thelevels of anti-VZV gE IgG in the serum of mice vaccinated with variousVZV gE mRNAs in comparison with VARIVAX® vaccine.

FIG. 6 is a graph showing the results of an ELISA assay indicating thelevels of anti-VZV gE IgG in the serum of mice vaccinated with variousVZV gE mRNAs in comparison with VARIVAX® vaccine.

FIG. 7 is a graph showing the results of an ELISA assay indicating thelevels of anti-VZV gE IgG in the serum of mice vaccinated with variousVZV gE mRNAs in comparison with VARIVAX® vaccine.

FIGS. 8A and 8B show confocal microscopy of human melanoma (MeWo) cellsstained with an antibodies to show the golgi apparatus.

FIGS. 9A-9C show confocal microscopy of MeWo cells stained withantibodies against VZV gE to show VZV gE expression and trafficking. Thesequence AEAADA depicted in FIG. 9C is SEQ ID NO: 58.

FIGS. 10A and 10B are schematics depicting various VZV wildtypegenotypes.

FIGS. 11A and 11B are graphs showing the results of an ELISA assay,which shows the levels of anti-VZV gE IgG in the serum of micevaccinated with VZV gE variant mRNAs in comparison with ZOSTAVAX®vaccine. The sequence AEAADA depicted throughout FIGS. 11A and 11B isSEQ ID NO: 58.

FIGS. 12A and 12B are graphs showing the results of an ELISA assay,which shows the levels of anti-VZV gE IgG in the serum of micevaccinated with VZV gE variant mRNAs in comparison with ZOSTAVAX®vaccine. The sequence AEAADA depicted throughout FIGS. 12A and 12B isSEQ ID NO: 58.

FIG. 13 is a graph showing the results of an ELISA assay measuring theantibody titer in the sera of mice immunized with VZV gE variant mRNAvaccines. Anti-VZV gE response induced by VZV gE variants mRNAs in miceare greater than that of ZOSTAVAX®. The gE variant mRNA forGE-delete_from_574-Y569A induced an immune response that isi log greaterthan ZOSTAVAX®. The sequence AEAADA depicted throughout FIG. 13 is SEQID NO: 58.

FIG. 14A is a graph showing the results of an ELISA assay, which showsthe levels of anti-VZV gE IgG in the serum of mice vaccinated with VZVgE variant mRNAs after primary exposure with ZOSTAVAX® vaccine (groups1-5) or VZV-gE-del_574_Y569A (group 6). The sequence AEAADA depictedthroughout FIG. 14A is SEQ ID NO: 58. FIG. 14B is a graph showing thedetermination of EC50.

FIGS. 15A and 15B are graphs showing the results of the T cell analysisof mice vaccinated with VZV gE variant mRNAs after primary exposure withZOSTAVAX® vaccine (groups 1-5) or VZV-gE-del_574_Y569A (group 6). Thesequence AEAADA depicted throughout FIGS. 15A and 15B is SEQ ID NO: 58.

FIGS. 16A and 16B are graphs showing the results of an ELISA assay,which shows the levels of anti-VZV gE IgG in serum of rhesus monkeysvaccinated with either VZV-gE-del_574_Y569A or ZOSTAVAX® after primaryexposure with VZV-gE-del_574_Y569A or ZOSTAVAX. FIG. 16C is a graphshowing the determination of EC50 and EC10. FIGS. 16D, 16E, and 16F aregraphs showing the results of the T cell analysis of rhesus monkeysvaccinated with either VZV-gE-del_574_Y569A or ZOSTAVAX® after primaryexposure with VZV-gE-del_574_Y569A or ZOSTAVAX®.

FIG. 17 shows next generation sequencing (NGS) data of VZV mRNA vaccineconstruct encoding a modified VZV-gE-del_574_Y569A antigen(VZV-gE-del_574_Y569A-v7). Compared to the VZV-gE-del_574_Y569A mRNAvaccine construct, which encodes the gE antigen (e.g., described inFIGS. 13-16), VZV-gE-del_574_Y569A-v7 was modified (6 nucleotide changeswere introduced) to reduce the occurrence of indel-causing homopolymericstretches (stretches of 6 As, 5 As, or 4 As). The data shows thatVZV-gE-del_574_Y569A-v7 does not contain significant indels.

FIG. 18A shows the average frequency of FITC⁺ MeWo cells six hours posttransfection with VZV-gE-del_574_Y569A or VZV-gE-del_574_Y569A-v7,measured by fluorescence-activated cell sorting (FACS). FIG. 18B showsthe FITC geometric mean.

FIGS. 19A-19B are Western blots showing detection ofVZV-gE-del_574_Y569A antigens in the lysates of HeLa cells 16 hours posttransfection. Expression of the VZV-gE-del_574_Y569A antigen wasdetected in HeLa cells transfected with the VZV-gE_574_Y569A mRNAconstruct and in HeLa cells transfected with the VZV-gE-del_574 Y569A-v7mRNA construct.

FIGS. 20A-20B are graphs showing the expression (FIG. 20A) andlocalization (FIG. 20B) of the VZV-gE-del_574_Y569A antigen expressed inHeLa cells transfected with either the VZV-gE-del_574_Y569A mRNAconstruct or the VZV-gE-del_574_Y569A-v7 mRNA construct.

FIGS. 21A-21C are microscopy images showing the localization in HeLacells (FIG. 21) or MeWo cells (FIGS. 21B and 21C) of full length VZV-gEand VZV-gE-del_574_Y569A antigens encoded by the VZV-gE-del_574_Y569AmRNA construct or the VZV-gE-del_574_Y569A-v7 mRNA construct. The fulllength VZV gE antigen localized to the golgi, and VZV-gE-del_574_Y569Aantigen localized to the cell membrane.

DETAILED DESCRIPTION

Embodiments of the present disclosure provide RNA (e.g., mRNA) vaccinesthat include at least one RNA (e.g., mRNA) polynucleotide encoding avaricella zoster virus (VZV) antigen. There are at least five clades ofvaricella zoster virus (VZV). Clades 1 and 3 include European/NorthAmerican strains; clade 2 includes Asian strains, especially from Japan;and clade 5 appears to be based in India. Clade 4 includes some strainsfrom Europe, but its geographic origins need further clarification.Phylogenetic analysis of VZV genomic sequences resolves wild-typestrains into 9 genotypes (E1, E2, J, M1, M2, M3, M4, VIII and IX).Sequence analysis of 342 clinical varicella and zoster specimens from 18European countries identified the following distribution of VZVgenotypes: E1, 221 (65%); E2, 87 (25%); M1, 20 (6%); M2, 3 (1%); M4, and11 (3%). No M3 or J strains were observed. Of 165 clinical varicella andzoster isolates from Australia and New Zealand, 67 of 127 easternAustralian isolates were E1, 30 were E2, 16 were J, 10 were M1, and 4were M2; 25 of 38 New Zealand isolates were E1, 8 were E2, and 5 wereM1.

VZV is an alphaherpesvirus that exists as a spherical multilayeredstructure approximately 200 nm in diameter. The viral genome issurrounded by a protein capsid structure that is covered by an amorphouslayer of tegument proteins. These two structures are surrounded by alipid envelope that is studded with viral glycoproteins, each about 8 nmin length, that are displayed on the exterior of the virion, andencloses the 100 nm nucleocapsid which is comprised of 162 hexameric andpentameric capsomeres arranged in an icosahedral form. The tegument,which is comprised of virally-encoded proteins and enzymes, is locatedin the space between the nucleocapsid and the viral envelope. The viralenvelope is acquired from host cell membranes and contains viral-encodedglycoproteins.

The VZV genome is a single, linear, duplex DNA molecule of 124,884 basepairs having at least 70 open reading frames. The genome has 2predominant isomers, depending on the orientation of the S segment, P(prototype) and IS (inverted S), which are present with equal frequencyfor a total frequency of 90-95%. The L segment can also be invertedresulting in a total of four linear isomers (IL and ILS).

VZV is closely related to the herpes simplex viruses (HSV), sharing muchgenome homology. The VZV genome is the smallest of the humanherpesviruses and encodes at least 71 unique proteins (ORFO-ORF68) withthree more opening reading frames (ORF69-ORF71) that duplicate earlieropen reading frames (ORF64-62, respectively). Only a fraction of theencoded proteins form the structure of the virus particle. Among thoseproteins are nine glycoproteins: ORF5 (gK), ORF9A (gN), ORF14 (gC),ORF31 (gB), ORF37 (gH), ORF50 (gM), ORF60 (gL), ORF67 (gI), and ORF68(gE). The known envelope glycoproteins (gB, gC, gE, gH, gI, gK, gL, gN,and gM) correspond with those in HSV; however, there is no equivalent ofHSV gD. VZV also fails to produce the LAT (latency-associatedtranscripts) that play an important role in establishing HSV latency(herpes simplex virus). The encoded glycoproteins gE, gI, gB, gH, gK,gL, gC, gN, and gM function in different steps of the viral replicationcycle. The most abundant glycoprotein found in infected cells, as wellas in the mature virion, is glycoprotein E (gE, ORF 68), which is amajor component of the virion envelope and is essential for viralreplication. Glycoprotein I (gI, ORG 67) forms a complex with gE ininfected cells, which facilitates the endocytosis of both glycoproteinsand directs them to the trans-Golgi network (TGN) where the final viralenvelope is acquired. Glycoprotein I (gI) is required within the TGN forVZV envelopment and for efficient membrane fusion during VZVreplication. VZV gE and gI are found complexed together on the infectedhost cell surface. Glycoprotein B (ORF 31), which is the second mostprevalent glycoprotein and thought to play a role in virus entry, bindsto neutralizing antibodies. Glycoprotein H is thought to have a fusionfunction facilitating cell to cell spread of the virus. Antibodies togE, gB, and gH are prevalent after natural infection and followingvaccination and have been shown to neutralize viral activity in vitro.

Embodiments of the present disclosure provide RNA (e.g., mRNA) vaccinesthat include at least one polynucleotide encoding at least one VZVantigenic polypeptide. The VZV RNA vaccines provided herein may be usedto induce a balanced immune response, comprising both cellular andhumoral immunity, without many of the risks associated with DNA vaccinesand live attenuated vaccines. The various RNA (e.g., mRNA) vaccinesdisclosed herein produced an immune response in BALB/C mice, the resultsof which are discussed in detail in the Examples section. Specifically,RNA (e.g., mRNA) polynucleotide vaccines having an open reading frameencoding one or more of a variety of VZV antigens produced significantimmune response, relative to a traditional VZV vaccine (e.g. attenuatedVZV virus). The VZV RNA (e.g., mRNA) polynucleotide vaccines disclosedherein encoding either VZV gE or variant VZV gE demonstrated significantimmune response after two administrations when administeredintramuscularly (IM) or intradermally (ID). The VZV glycoproteins andtegument proteins have been shown to be antigenic. VZV glycoproteins,fragments thereof, and epitopes thereof are encompassed within thepresent disclosure.

The entire contents of International Application No. PCT/US2015/027400,International Publication No. WO2015/164674A, are incorporated herein byreference.

It has been discovered that the mRNA vaccines described herein aresuperior to current vaccines in several ways. First, the lipidnanoparticle (LNP) delivery is superior to other formulations includinga protamine base approach described in the literature and no additionaladjuvants are to be necessary. The use of LNPs enables the effectivedelivery of chemically modified or unmodified mRNA vaccines.Additionally it has been demonstrated herein that both modified andunmodified LNP formulated mRNA vaccines were superior to conventionalvaccines by a significant degree. In some embodiments the mRNA vaccinesof the invention are superior to conventional vaccines by a factor of atleast 10 fold, 20 fold, 40 fold, 50 fold, 100 fold, 500 fold or 1,000fold.

Although attempts have been made to produce functional RNA vaccines,including mRNA vaccines and self-replicating RNA vaccines, thetherapeutic efficacy of these RNA vaccines have not yet been fullyestablished. Quite surprisingly, the inventors have discovered,according to aspects of the invention a class of formulations fordelivering mRNA vaccines in vivo that results in significantly enhanced,and in many respects synergistic, immune responses including enhancedantigen generation and functional antibody production withneutralization capability. These results can be achieved even whensignificantly lower doses of the mRNA are administered in comparisonwith mRNA doses used in other classes of lipid based formulations. Theformulations of the invention have demonstrated significant unexpectedin vivo immune responses sufficient to establish the efficacy offunctional mRNA vaccines as prophylactic and therapeutic agents.Additionally, self-replicating RNA vaccines rely on viral replicationpathways to deliver enough RNA to a cell to produce an immunogenicresponse. The formulations of the invention do not require viralreplication to produce enough protein to result in a strong immuneresponse. Thus, the mRNA of the invention are not self-replicating RNAand do not include components necessary for viral replication.

The invention involves, in some aspects, the surprising finding thatlipid nanoparticle (LNP) formulations significantly enhance theeffectiveness of mRNA vaccines, including chemically modified andunmodified mRNA vaccines. The efficacy of mRNA vaccines formulated inLNP was examined in vivo using several distinct antigens. The resultspresented herein demonstrate the unexpected superior efficacy of themRNA vaccines formulated in LNP over other commercially availablevaccines.

In addition to providing an enhanced immune response, the formulationsof the invention generate a more rapid immune response with fewer dosesof antigen than other vaccines tested. The mRNA-LNP formulations of theinvention also produce quantitatively and qualitatively better immuneresponses than vaccines formulated in a different carriers.

The data described herein demonstrate that the formulations of theinvention produced significant unexpected improvements over existing VZVantigen vaccines, including significantly higher levels of IgGproduction by mRNA chemically modified and unmodified VZV vaccinesformulated in LNP compared to VARIVAX and ZOSTAVAX. The onset of IgGproduction was significantly more rapid for the chemically modified LNPmRNA vaccines than the unmodified or commercially available vaccinestested.

Additionally, the mRNA-LNP formulations of the invention are superior toother vaccines even when the dose of mRNA is lower than other vaccines.The data demonstrate that all gE variants LNP mRNA vaccines induced muchstronger immune response than ZOSTAVAX® after the two 10 μg doses aswell as after the two 2 μg doses. When the sera were diluted more than100 fold, the antibody titer is higher in VZV gE LNP mRNA vaccinatedmice sera than in ZOSTAVAX® vaccinated mice sera, suggesting that theVZV gE LNP mRNA vaccines induced much stronger immune response thanZOSTAVAX® in mice.

The results in mice were consistent with the immunogenicity observed innon-human primates. Rhesus monkeys were primed with chemically modifiedVZV LNP mRNA vaccines or ZOSTAVAX®. The mRNA vaccines provided higheranti-gE titers than ZOSTAVAX® and produced reasonable frequency of CD4T-cells producing IFNγ, IL-2 or TNFα cells, unlike the ZOSTAVAX® group.The data also demonstrated that a single dose of mRNA vaccination afterZOSTAVAX® exposure was equivalent to two doses of mRNA vaccination ininducing comparable T-cell responses.

Some of the LNP used in the studies described herein has been usedpreviously to deliver siRNA in various animal models as well as inhumans. In view of the observations made in association with the siRNAdelivery of LNP formulations, the fact that LNP is useful in vaccines isquite surprising. It has been observed that therapeutic delivery ofsiRNA formulated in LNP causes an undesirable inflammatory responseassociated with a transient IgM response, typically leading to areduction in antigen production and a compromised immune response. Incontrast to the findings observed with siRNA, the LNP-mRNA formulationsof the invention are demonstrated herein to generate enhanced IgGlevels, sufficient for prophylactic and therapeutic methods rather thantransient IgM responses.

Antigens/Antigenic Polypeptides

In some embodiments, an antigenic polypeptide is a VZV glycoprotein. Forexample, a VZV glycoprotein may be VZV gE, gI, gB, gH, gK, gL, gC, gN,or gM. In some embodiments, the antigenic polypeptide is a VZV gEpolypeptide. In some embodiments, the antigenic polypeptide is a VZV gIpolypeptide. In some embodiments, the antigenic polypeptide is a VZV gBpolypeptide. In some embodiments, the antigenic polypeptide is a VZV gHpolypeptide. In some embodiments, the antigenic polypeptide is a VZV gKpolypeptide. In some embodiments, the antigenic polypeptide is a VZV gLpolypeptide. In some embodiments, the antigenic polypeptide is a VZV gCpolypeptide. In some embodiments, the antigenic polypeptide is a VZV gNpolypeptide. In some embodiments, the antigenic polypeptide is a VZV gMpolypeptide.

In some embodiments, the antigenic polypeptide comprises two or moreglycoproteins. The two or more glycoproteins can be encoded by a singleRNA polynucleotide or can be encoded by two or more RNA polynucleotides,for example, each glycoprotein encoded by a separate RNA polynucleotide.In some embodiments, the two or more glycoproteins can be anycombination of VZV gE, gI, gB, gH, gK, gL, gC, gN, and gM polypeptides.In some embodiments, the two or more glycoproteins can be anycombination of VZV gE and a glycoprotein selected from gI, gB, gH, gK,gL, gC, gN, and gM polypeptides. In some embodiments, the two or moreglycoproteins can be any combination of VZV gI and a glycoproteinselected from gE, gB, gH, gK, gL, gC, gN, and gM polypeptides. In someembodiments, the two or more glycoproteins can be any combination of VZVgE, gI, and a glycoprotein selected from gB, gH, gK, gL, gC, gN, and gMpolypeptides. In some embodiments, the two or more VZV glycoproteins aregE and gI. Alternate RNA vaccines comprising RNA polynucleotidesencoding other viral protein components of VZV, for example, tegumentproteins are encompassed by the present disclosure. Thus, someembodiments of the present disclosure provide VZV vaccines that includeat least one ribonucleic acid (RNA) polynucleotide having an openreading frame encoding at least one VZV tegument protein. In someembodiments, the antigenic polypeptide is a VZV tegument protein. Inother embodiments, the antigenic fragment(s) of the VZV vaccine may beat least one VZV tegument polypeptide and at least one VZV glycoproteinpolypeptide, for example any VZV glycoprotein selected from gE, gI, gB,gH, gK, gL, gC, gN, and gM.

The present disclosure includes variant VZV antigenic polypeptides. Insome embodiments, the variant VZV antigenic polypeptide is a variant VZVgE polypeptide. The variant VZV gE polypeptides are designed to avoidER/golgi retention of polypeptides, leading to increased surfaceexpression of the antigen. In some embodiments, the variant gEpolypeptides are truncated to remove the ER retention portion or thecytoplasmic tail portion of the polypeptide. In some embodiments, thevariant VZV gE polypeptides are mutated to reduce VZV polypeptidelocalization to the ER/golgi/TGN. Such modifications inhibit ER trappingand, as such, expedite trafficking to the cell membrane.

Thus, in some embodiments, the VZV glycoprotein is a variant gEpolypeptide. VZV gE has targeting sequences for the TGN in itsC-terminus and is transported from the ER to the TGN in infected andgE-transfected cells. Most gE in the TGN appears to be retrieved byendocytosis from the plasma membrane and delivered to the TGN byendosomes, which is followed by recycling to the plasma membranes. gE isaccumulated in TGN, along with other VZV proteins (e.g., tegumentproteins) associated with the production of fully enveloped VZV virions.Thus, mutations to reduce TGN localization and endocytosis aids in thetrafficking of gE to the cell membrane.

The variant VZV gE polypeptide can be any truncated polypeptide lackingthe anchor domain (ER retention domain). For example, the variant VZV gEpolypeptide can be a truncated VZV gE polypeptide comprising at leastamino acids 1-124, including, for example, amino acids 1-124, 1-140,1-160, 1-200, 1-250, 1-300, 1-350, 1-360, 1-400, 1-450, 1-500, 1-511,1-550, and 1-561, as well as polypeptide fragments having fragment sizeswithin the recited size ranges. In one embodiment, the truncated VZV gEpolypeptide comprises amino acids 1-561 of SEQ ID NO: 10. In someembodiments, the variant VZV gE polypeptide is a truncated polypeptidelacking the carboxy terminal tail domain. Thus in some embodiments, thetruncated VZV gE polypeptide comprises amino acids 1-573 of SEQ ID NO:10.

In some embodiments, the variant VZV gE polypeptide has at least onemutation in one or more motif(s) associated with ER retention, whereinthe mutation(s) in one or more motif(s) results in decreased retentionof the VZV gE polypeptide in the ER and/or golgi. In some embodiments,the variant VZV gE polypeptide has at least one mutation in one or morephosphorylated acidic motif(s). For example, the variant VZV gEpolypeptide can be a full-length VZV gE polypeptide having a Y582Gmutation, a Y569A mutation, or both a Y582G mutation and a Y569Amutation. Alternatively, the variant VZV gE polypeptide can be anantigenic fragment comprising, for example, amino acids 1-573 of VZV gEand having a Y569A mutation. Alternatively, the variant VZV gEpolypeptide can be an antigenic fragment having mutation in an acidicphosphorylation motif, such as an SST motif. For example, the variantVZV gE polypeptide can be an antigenic fragment having AEAADA sequence(SEQ ID NO: 58).

In some embodiments, the variant VZV gE polypeptide is a full-length VZVgE polypeptide having additional sequence at the C-terminus which aidsin secretion of the polypeptide. For example, the variant VZV gEpolypeptide can be a full-length VZV gE polypeptide having an IgKappasequence at the C-terminus. In some embodiments, the VZV gE polypeptidehas additional sequence at the C-terminus that aids in secretion (I., anIgKappa sequence at the C-terminus) and the variant VZV gE polypeptidehas at least one mutation in one or more motif(s) associated with ERretention, TGN localization, and/or endocytosis (e.g., a Y582G mutation,a Y569A mutation, or both a Y582G mutation and a Y569A mutation) and/orat least one mutation in one or more phosphorylated acidic motif(s). Insome embodiments, the variant VZV gE polypeptide is a truncatedpolypeptide lacking the anchor domain (ER retention domain) and havingan additional sequence at the C-terminus which aids in secretion of thepolypeptide, for example, an IgKappa sequence at the C-terminus. In someembodiments, the truncated VZV gE polypeptide comprises amino acids1-561 of SEQ ID NO: 10 and has an IgKappa sequence at the C-terminus. Insome embodiments, the variant VZV gE polypeptide is a truncatedpolypeptide lacking the carboxy terminal tail domain and having anadditional sequence at the C-terminus that aids in secretion of thepolypeptide (e.g., having an IgKappa sequence at the C-terminus). Insome embodiments, the truncated VZV gE polypeptide comprises amino acids1-573 of SEQ ID NO: 10 and has an IgKappa sequence at the C-terminus.

In some embodiments, a VZV antigenic polypeptide is longer than 25 aminoacids and shorter than 50 amino acids. The term “antigenic polypeptide”and “antigenic protein” includes immunogenic fragments and epitopesthereof. Thus, polypeptides include gene products, naturally occurringpolypeptides, synthetic polypeptides, homologs, orthologs, paralogs,fragments and other equivalents, variants, and analogs of the foregoing.A polypeptide may be a single molecule or may be a multi-molecularcomplex such as a dimer, trimer or tetramer. Polypeptides may alsocomprise single chain or multichain polypeptides such as antibodies orinsulin and may be associated or linked. Most commonly, disulfidelinkages are found in multichain polypeptides. The term polypeptide mayalso apply to amino acid polymers in which at least one amino acidresidue is an artificial chemical analogue of a correspondingnaturally-occurring amino acid.

The term “polypeptide variant” refers to molecules which differ in theiramino acid sequence from a native or reference sequence. The amino acidsequence variants may possess substitutions, deletions, and/orinsertions at certain positions within the amino acid sequence, ascompared to a native or reference sequence. Ordinarily, variants possessat least 50% identity to a native or reference sequence. In someembodiments, variants share at least 80%, or at least 90% identity witha native or reference sequence.

In some embodiments “variant mimics” are provided. As used herein, a“variant mimic” contains at least one amino acid that would mimic anactivated sequence. For example, glutamate may serve as a mimic forphosphoro-threonine and/or phosphoro-serine.

Alternatively, variant mimics may result in deactivation or in aninactivated product containing the mimic. For example, phenylalanine mayact as an inactivating substitution for tyrosine, or alanine may act asan inactivating substitution for serine.

“Orthologs” refers to genes in different species that evolved from acommon ancestral gene by speciation. Normally, orthologs retain the samefunction in the course of evolution. Identification of orthologs iscritical for reliable prediction of gene function in newly sequencedgenomes.

“Analogs” is meant to include polypeptide variants that differ by one ormore amino acid alterations, for example, substitutions, additions ordeletions of amino acid residues that still maintain one or more of theproperties of the parent or starting polypeptide.

“Paralogs” are genes (or proteins) related by duplication within agenome. Orthologs retain the same function in the course of evolution,whereas paralogs evolve new functions, even if these are related to theoriginal one.

The present disclosure provides several types of compositions that arepolynucleotide or polypeptide based, including variants and derivatives.These include, for example, substitutional, insertional, deletion andcovalent variants and derivatives. The term “derivative” is usedsynonymously with the term “variant,” but generally refers to a moleculethat has been modified and/or changed in any way relative to a referencemolecule or starting molecule.

As such, polynucleotides encoding peptides or polypeptides containingsubstitutions, insertions and/or additions, deletions and covalentmodifications with respect to reference sequences, in particular thepolypeptide sequences disclosed herein, are included within the scope ofthis disclosure. For example, sequence tags or amino acids, such as oneor more lysines, can be added to peptide sequences (e.g., at theN-terminal or C-terminal ends). Sequence tags can be used for peptidedetection, purification or localization. Lysines can be used to increasepeptide solubility or to allow for biotinylation. Alternatively, aminoacid residues located at the carboxy and amino terminal regions of theamino acid sequence of a peptide or protein may optionally be deletedproviding for truncated sequences. Certain amino acids (e.g., C-terminalor N-terminal residues) may alternatively be deleted depending on theuse of the sequence, as for example, expression of the sequence as partof a larger sequence which is soluble, or linked to a solid support. Inalternative embodiments, sequences for (or encoding) signal sequences,termination sequences, transmembrane domains, linkers, multimerizationdomains (such as, e.g., foldon regions) and the like may be substitutedwith alternative sequences that achieve the same or a similar function.Such sequences are readily identifiable to one of skill in the art. Itshould also be understood that some of the sequences provided hereincontain sequence tags or terminal peptide sequences (e.g., at theN-terminal or C-terminal ends) that may be deleted, for example, priorto use in the preparation of an RNA (e.g., mRNA) vaccine.

“Substitutional variants” when referring to polypeptides are those thathave at least one amino acid residue in a native or starting sequenceremoved and a different amino acid inserted in its place at the sameposition. Substitutions may be single, where only one amino acid in themolecule has been substituted, or they may be multiple, where two ormore amino acids have been substituted in the same molecule.

As used herein the term “conservative amino acid substitution” refers tothe substitution of an amino acid that is normally present in thesequence with a different amino acid of similar size, charge, orpolarity. Examples of conservative substitutions include thesubstitution of a non-polar (hydrophobic) residue such as isoleucine,valine and leucine for another non-polar residue. Likewise, examples ofconservative substitutions include the substitution of one polar(hydrophilic) residue for another such as between arginine and lysine,between glutamine and asparagine, and between glycine and serine.Additionally, the substitution of a basic residue, such as lysine,arginine or histidine for another, or the substitution of one acidicresidue, such as aspartic acid or glutamic acid for another acidicresidue are additional examples of conservative substitutions. Examplesof non-conservative substitutions include the substitution of anon-polar (hydrophobic) amino acid residue such as isoleucine, valine,leucine, alanine, methionine for a polar (hydrophilic) residue such ascysteine, glutamine, glutamic acid or lysine and/or a polar residue fora non-polar residue.

“Features” when referring to polypeptide or polynucleotide are definedas distinct amino acid sequence-based or nucleotide-based components ofa molecule respectively. Features of the polypeptides encoded by thepolynucleotides include surface manifestations, local conformationalshape, folds, loops, half-loops, domains, half-domains, sites, terminior any combination thereof.

As used herein when referring to polypeptides the term “domain” refersto a motif of a polypeptide having one or more identifiable structuralor functional characteristics or properties (e.g., binding capacity,serving as a site for protein-protein interactions).

As used herein, when referring to polypeptides the terms “site” as itpertains to amino acid based embodiments, is used synonymously with“amino acid residue” and “amino acid side chain.” As used herein, whenreferring to polynucleotides the terms “site” as it pertains tonucleotide based embodiments, is used synonymously with “nucleotide.” Asite represents a position within a peptide or polypeptide orpolynucleotide that may be modified, manipulated, altered, derivatizedor varied within the polypeptide or polynucleotide based molecules.

As used herein, the terms “termini” or “terminus,” when referring topolypeptides or polynucleotides, refers to an extremity of a polypeptideor polynucleotide respectively. Such extremity is not limited only tothe first or final site of the polypeptide or polynucleotide but mayinclude additional amino acids or nucleotides in the terminal regions.Polypeptide-based molecules may be characterized as having both anN-terminus (terminated by an amino acid with a free amino group (NH2))and a C-terminus (terminated by an amino acid with a free carboxyl group(COOH)). Proteins are in some cases made up of multiple polypeptidechains brought together by disulfide bonds or by non-covalent forces(multimers, oligomers). These proteins have multiple N-termini andC-termini. Alternatively, the termini of the polypeptides may bemodified such that they begin or end, as the case may be, with anon-polypeptide based moiety such as an organic conjugate.

As recognized by those skilled in the art, protein fragments, functionalprotein domains, and homologous proteins are also considered to bewithin the scope of polypeptides of interest. For example, providedherein is any protein fragment (meaning a polypeptide sequence at leastone amino acid residue shorter than a reference polypeptide sequence butotherwise identical) of a reference protein 10, 20, 30, 40, 50, 60, 70,80, 90, 100 or greater than 100 amino acids in length. In anotherexample, any protein that includes a stretch of 20, 30, 40, 50, or 100amino acids that are 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100%identical to any of the sequences described herein can be utilized inaccordance with the present disclosure. In some embodiments, apolypeptide includes 2, 3, 4, 5, 6, 7, 8, 9, 10, or more mutations, asshown in any of the sequences provided or referenced herein. In someembodiments, a protein fragment is longer than 25 amino acids andshorter than 50 amino acids.

Polypeptide or polynucleotide molecules of the present disclosure mayshare a certain degree of sequence similarity or identity with thereference molecules (e.g., reference polypeptides or referencepolynucleotides), for example, with art-described molecules (e.g.,engineered or designed molecules or wild-type molecules). The term“identity,” as known in the art, refers to a relationship between thesequences of two or more polypeptides or polynucleotides, as determinedby comparing the sequences. In the art, identity also means the degreeof sequence relatedness between them as determined by the number ofmatches between strings of two or more amino acid residues or nucleicacid residues. Identity measures the percent of identical matchesbetween the smaller of two or more sequences with gap alignments (ifany) addressed by a particular mathematical model or computer program(e.g., “algorithms”). Identity of related peptides can be readilycalculated by known methods. “% identity” as it applies to polypeptideor polynucleotide sequences is defined as the percentage of residues(amino acid residues or nucleic acid residues) in the candidate aminoacid or nucleic acid sequence that are identical with the residues inthe amino acid sequence or nucleic acid sequence of a second sequenceafter aligning the sequences and introducing gaps, if necessary, toachieve the maximum percent identity. Methods and computer programs forthe alignment are well known in the art. It is understood that identitydepends on a calculation of percent identity but may differ in value dueto gaps and penalties introduced in the calculation. Generally, variantsof a particular polynucleotide or polypeptide have at least 40%, 45%,50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98%, 99% but less than 100% sequence identity to thatparticular reference polynucleotide or polypeptide as determined bysequence alignment programs and parameters described herein and known tothose skilled in the art. Such tools for alignment include those of theBLAST suite (Stephen F. Altschul, et al (1997), “Gapped BLAST andPSI-BLAST: a new generation of protein database search programs”,Nucleic Acids Res. 25:3389-3402). Another popular local alignmenttechnique is based on the Smith-Waterman algorithm (Smith, T. F. &Waterman, M. S. (1981) “Identification of common molecularsubsequences.” J. Mol. Biol. 147:195-197). A general global alignmenttechnique based on dynamic programming is the Needleman-Wunsch algorithm(Needleman, S. B. & Wunsch, C. D. (1970) “A general method applicable tothe search for similarities in the amino acid sequences of twoproteins.” J. Mol. Biol. 48:443-453). More recently a Fast OptimalGlobal Sequence Alignment Algorithm (FOGSAA) has been developed thatpurportedly produces global alignment of nucleotide and proteinsequences faster than other optimal global alignment methods, includingthe Needleman-Wunsch algorithm. Other tools are described herein,specifically in the definition of “identity” below.

As used herein, the term “homology” refers to the overall relatednessbetween polymeric molecules, e.g. between nucleic acid molecules (e.g.DNA molecules and/or RNA molecules) and/or between polypeptidemolecules. Polymeric molecules (e.g. nucleic acid molecules (e.g. DNAmolecules and/or RNA molecules) and/or polypeptide molecules) that sharea threshold level of similarity or identity determined by alignment ofmatching residues are termed homologous. Homology is a qualitative termthat describes a relationship between molecules and can be based uponthe quantitative similarity or identity. Similarity or identity is aquantitative term that defines the degree of sequence match between twocompared sequences. In some embodiments, polymeric molecules areconsidered to be “homologous” to one another if their sequences are atleast 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%,90%, 95%, or 99% identical or similar. The term “homologous” necessarilyrefers to a comparison between at least two sequences (polynucleotide orpolypeptide sequences). Two polynucleotide sequences are consideredhomologous if the polypeptides they encode are at least 50%, 60%, 70%,80%, 90%, 95%, or even 99% for at least one stretch of at least 20 aminoacids. In some embodiments, homologous polynucleotide sequences arecharacterized by the ability to encode a stretch of at least 4-5uniquely specified amino acids. For polynucleotide sequences less than60 nucleotides in length, homology is determined by the ability toencode a stretch of at least 4-5 uniquely specified amino acids. Twoprotein sequences are considered homologous if the proteins are at least50%, 60%, 70%, 80%, or 90% identical for at least one stretch of atleast 20 amino acids.

Homology implies that the compared sequences diverged in evolution froma common origin. The term “homolog” refers to a first amino acidsequence or nucleic acid sequence (e.g., gene (DNA or RNA) or proteinsequence) that is related to a second amino acid sequence or nucleicacid sequence by descent from a common ancestral sequence. The term“homolog” may apply to the relationship between genes and/or proteinsseparated by the event of speciation or to the relationship betweengenes and/or proteins separated by the event of genetic duplication.

Nucleic Acids/Polynucleotides

Varicella zoster virus (VZV) vaccines, as provided herein, comprise atleast one (one or more) ribonucleic acid (RNA, e.g., mRNA)polynucleotide having an open reading frame encoding at least one VZVantigenic polypeptide. The term “nucleic acid,” in its broadest sense,includes any compound and/or substance that comprises a polymer ofnucleotides. These polymers are referred to as polynucleotides.

In some embodiments, at least one RNA polynucleotide of a VZV vaccine isencoded by at least one nucleic acid sequence selected from SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6,SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 41.

In some embodiments, at least one RNA (e.g., mRNA) polynucleotide of aVZV vaccine is encoded by at least one fragment of a nucleic acidsequence (e.g., a fragment having an antigenic sequence or at least oneepitope) selected from SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ IDNO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQID NO: 41.

Nucleic acids (also referred to as polynucleotides) may be or mayinclude, for example, ribonucleic acids (RNAs), deoxyribonucleic acids(DNAs), threose nucleic acids (TNAs), glycol nucleic acids (GNAs),peptide nucleic acids (PNAs), locked nucleic acids (LNAs, including LNAhaving a βD-ribo configuration, α-LNA having an α-L-ribo configuration(a diastereomer of LNA), 2′-amino-LNA having a 2′-aminofunctionalization, and 2′-amino-α-LNA having a 2′-aminofunctionalization), ethylene nucleic acids (ENA), cyclohexenyl nucleicacids (CeNA) or chimeras or combinations thereof.

In some embodiments, polynucleotides of the present disclosure functionas messenger RNA (mRNA). “Messenger RNA” (mRNA) refers to anypolynucleotide that encodes a (at least one) polypeptide (anaturally-occurring, non-naturally-occurring, or modified polymer ofamino acids) and can be translated to produce the encoded polypeptide invitro, in vivo, in situ or ex vivo. The skilled artisan will appreciatethat, except where otherwise noted, polynucleotide sequences set forthin the instant application will recite “T”s in a representative DNAsequence, but where the sequence represents RNA (e.g., mRNA), the “T”swould be substituted for “U”s. Thus, any of the RNA polynucleotidesencoded by a DNA identified by a particular sequence identificationnumber may also comprise the corresponding RNA (e.g., mRNA) sequenceencoded by the DNA, where each “T” of the DNA sequence is substitutedwith “U.” It should be understood that the mRNA polynucleotides of thevaccines as provided herein are synthetic molecules, i.e., they are notnaturally-occurring molecules. That is, the mRNA polynucleotides of thepresent disclosure are isolated mRNA polynucleotides. As is known in theart, “isolated polynucleotides” refer to polynucleotides that aresubstantially physically separated from other cellular material (e.g.,separated from cells and/or systems that produce the polynucleotides) orfrom other material that hinders their use in the vaccines of thepresent disclosure. Isolated polynucleotides are substantially pure inthat they have been substantially separated from the substances withwhich they may be associated in living or viral systems. Thus, mRNApolynucleotide vaccines are not associated with living or viral systems,such as cells or viruses. The mRNA polynucleotide vaccines do notinclude viral components (e.g., viral capsids, viral enzymes, or otherviral proteins, for example, those needed for viral-based replication),and the mRNA polynucleotide vaccines are not packaged within,encapsulated within, linked to, or otherwise associated with a virus orviral particle. In some embodiments, the mRNA vaccines comprise a lipidnanoparticle that consists of, or consists essentially of, one or moremRNA polynucleotides (e.g., mRNA polynucleotides encoding one or moreVZV antigen(s)).

The basic components of an mRNA molecule typically include at least onecoding region, a 5′ untranslated region (UTR), a 3′ UTR, a 5′ cap and apoly-A tail. Polynucleotides of the present disclosure may function asmRNA but can be distinguished from wild-type mRNA in their functionaland/or structural design features, which serve to overcome existingproblems of effective polypeptide expression using nucleic-acid basedtherapeutics. In some embodiments, the RNA is a messenger RNA (mRNA)having an open reading frame encoding at least one VZV antigen. In someembodiments, the RNA (e.g., mRNA) further comprises a (at least one) 5′UTR, 3′ UTR, a polyA tail and/or a 5′ cap.

In some embodiments, a RNA polynucleotide (e.g., mRNA) of a VZV vaccineencodes 2-10, 2-9, 2-8, 2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 3-9, 3-8, 3-7,3-6, 3-5, 3-4, 4-10, 4-9, 4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5-6,6-10, 6-9, 6-8, 6-7, 7-10, 7-9, 7-8, 8-10, 8-9 or 9-10 antigenicpolypeptides. In some embodiments, a RNA polynucleotide (e.g., mRNA) ofa VZV RNA (e.g., mRNA) vaccine encodes at least 10, 20, 30, 40, 50, 60,70, 80, 90 or 100 antigenic polypeptides. In some embodiments, a RNApolynucleotide (e.g., mRNA) of a VZV vaccine encodes at least 100antigenic polypeptides, or at least 200 antigenic polypeptides. In someembodiments, a RNA polynucleotide (e.g., mRNA) of a VZV vaccine encodes1-10, 5-15, 10-20, 15-25, 20-30, 25-35, 30-40, 35-45, 40-50, 1-50,1-100, 2-50 or 2-100 antigenic polypeptides.

Polynucleotides (e.g., mRNAs) of the present disclosure, in someembodiments, are codon optimized. Codon optimization methods are knownin the art and may be used as provided herein. For example, any one ormore of the sequences SEQ ID NO: 11, 15, 19, 23, 27, 31, 35, 39, 62, 66,70, 74, 78, 82, 86, 90 or any one or more of the sequences of SEQ ID NO:92-108 may be codon optimized. Codon optimization, in some embodiments,may be used to match codon frequencies in target and host organisms toensure proper folding; bias GC content to increase mRNA stability orreduce secondary structures; minimize tandem repeat codons or base runsthat may impair gene construction or expression; customizetranscriptional and translational control regions; insert or removeprotein trafficking sequences; remove/add post translation modificationsites in encoded protein (e.g., glycosylation sites); add, remove orshuffle protein domains; insert or delete restriction sites; modifyribosome binding sites and mRNA degradation sites; adjust translationalrates to allow the various domains of the protein to fold properly; orreduce or eliminate problem secondary structures within thepolynucleotide. Codon optimization tools, algorithms and services areknown in the art—non-limiting examples include services from GeneArt(Life Technologies), DNA2.0 (Menlo Park Calif.) and/or proprietarymethods. In some embodiments, the open reading frame (ORF) sequence isoptimized using optimization algorithms.

In some embodiments, a codon optimized sequence (e.g., a codon-optimizedsequence any one of SEQ ID NO: 92-108) shares less than 95% sequenceidentity to a naturally-occurring or wild-type sequence (e.g., anaturally-occurring or wild-type mRNA sequence encoding a polypeptide orprotein of interest (e.g., an antigenic protein or polypeptide)). Insome embodiments, a codon optimized sequence shares less than 90%sequence identity to a naturally-occurring or wild-type sequence (e.g.,a naturally-occurring or wild-type mRNA sequence encoding a polypeptideor protein of interest (e.g., an antigenic protein or polypeptide)). Insome embodiments, a codon optimized sequence shares less than 85%sequence identity to a naturally-occurring or wild-type sequence (e.g.,a naturally-occurring or wild-type mRNA sequence encoding a polypeptideor protein of interest (e.g., an antigenic protein or polypeptide)). Insome embodiments, a codon optimized sequence shares less than 80%sequence identity to a naturally-occurring or wild-type sequence (e.g.,a naturally-occurring or wild-type mRNA sequence encoding a polypeptideor protein of interest (e.g., an antigenic protein or polypeptide)). Insome embodiments, a codon optimized sequence shares less than 75%sequence identity to a naturally-occurring or wild-type sequence (e.g.,a naturally-occurring or wild-type mRNA sequence encoding a polypeptideor protein of interest (e.g., an antigenic protein or polypeptide)).

In some embodiments, a codon optimized sequence shares between 65% and85% (e.g., between about 67% and about 85% or between about 67% andabout 80%) sequence identity to a naturally-occurring or wild-typesequence (e.g., a naturally-occurring or wild-type mRNA sequenceencoding a polypeptide or protein of interest (e.g., an antigenicprotein or polypeptide)). In some embodiments, a codon optimizedsequence shares between 65% and 75% or about 80% sequence identity to anaturally-occurring or wild-type sequence (e.g., a naturally-occurringor wild-type mRNA sequence encoding a polypeptide or protein of interest(e.g., an antigenic protein or polypeptide)).

In some embodiments, a codon-optimized sequence (e.g., a codon-optimizedsequence of any one of SEQ ID NO: 92-108) encodes an antigenicpolypeptide that is as immunogenic as, or more immunogenic than (e.g.,at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 100%, or at least 200% more), than an antigenic polypeptideencoded by a (non-codon-optimized) sequence of any one of SEQ ID NO:92-108.

In some embodiments, the VZV vaccine includes at least one RNApolynucleotide having an open reading frame encoding at least one VZVantigenic polypeptide having at least one modification, at least one 5′terminal cap, and is formulated within a lipid nanoparticle. 5′-cappingof polynucleotides may be completed concomitantly during the invitro-transcription reaction using the following chemical RNA capanalogs to generate the 5′-guanosine cap structure according tomanufacturer protocols: 3′-O-Me-m7G(5′)ppp(5′) G [the ARCA cap];G(5′)ppp(5′)A; G(5′)ppp(5′)G; m7G(5′)ppp(5′)A; m7G(5′)ppp(5′)G (NewEngland BioLabs, Ipswich, Mass.). 5′-capping of modified RNA may becompleted post-transcriptionally using a Vaccinia Virus Capping Enzymeto generate the “Cap 0” structure: m7G(5′)ppp(5′)G (New England BioLabs,Ipswich, Mass.). Cap 1 structure may be generated using both VacciniaVirus Capping Enzyme and a 2′-O methyl-transferase to generate:m7G(5′)ppp(5′)G-2′-O-methyl. Cap 2 structure may be generated from theCap 1 structure followed by the 2′-O-methylation of the5′-antepenultimate nucleotide using a 2′-O methyl-transferase. Cap 3structure may be generated from the Cap 2 structure followed by the2′-O-methylation of the 5′-preantepenultimate nucleotide using a 2′-Omethyl-transferase. Enzymes may be derived from a recombinant source.

When transfected into mammalian cells, the modified mRNAs have astability of between 12-18 hours, or greater than 18 hours, e.g., 24,36, 48, 60, 72, or greater than 72 hours.

In some embodiments a codon optimized RNA may be one in which the levelsof G/C are enhanced. The G/C-content of nucleic acid molecules (e.g.,mRNA) may influence the stability of the RNA. RNA having an increasedamount of guanine (G) and/or cytosine (C) residues may be functionallymore stable than RNA containing a large amount of adenine (A) andthymine (T) or uracil (U) nucleotides. As an example, WO2002/098443discloses a pharmaceutical composition containing an mRNA stabilized bysequence modifications in the translated region. Due to the degeneracyof the genetic code, the modifications work by substituting existingcodons for those that promote greater RNA stability without changing theresulting amino acid. The approach is limited to coding regions of theRNA.

Signal Peptides

In some embodiments, antigenic polypeptides encoded by VZVpolynucleotides comprise a signal peptide. Signal peptides, comprisingthe N-terminal 15-60 amino acids of proteins, are typically needed forthe translocation across the membrane on the secretory pathway and thusuniversally control the entry of most proteins both in eukaryotes andprokaryotes to the secretory pathway. Signal peptides generally includeof three regions: an N-terminal region of differing length, whichusually comprises positively charged amino acids; a hydrophobic region;and a short carboxy-terminal peptide region. In eukaryotes, the signalpeptide of a nascent precursor protein (pre-protein) directs theribosome to the rough endoplasmic reticulum (ER) membrane and initiatesthe transport of the growing peptide chain across it. The signal peptideis not responsible for the final destination of the mature protein,however. Secretory proteins devoid of further address tags in theirsequence are by default secreted to the external environment. Signalpeptides are cleaved from precursor proteins by an endoplasmic reticulum(ER)-resident signal peptidase or they remain uncleaved and function asa membrane anchor. During recent years, a more advanced view of signalpeptides has evolved, showing that the functions and immunodominance ofcertain signal peptides are much more versatile than previouslyanticipated.

Signal peptides typically function to facilitate the targeting of newlysynthesized protein to the endoplasmic reticulum (ER) for processing. ERprocessing produces a mature Envelope protein, wherein the signalpeptide is cleaved, typically by a signal peptidase of the host cell. Asignal peptide may also facilitate the targeting of the protein to thecell membrane. VZV vaccines of the present disclosure may comprise, forexample, RNA polynucleotides encoding an artificial signal peptide,wherein the signal peptide coding sequence is operably linked to and isin frame with the coding sequence of the VZV antigenic polypeptide.Thus, VZV vaccines of the present disclosure, in some embodiments,produce an antigenic polypeptide comprising a VZV antigenic polypeptidefused to a signal peptide. In some embodiments, a signal peptide isfused to the N-terminus of the VZV antigenic polypeptide. In someembodiments, a signal peptide is fused to the C-terminus of the VZVantigenic polypeptide.

In some embodiments, the signal peptide fused to the VZV antigenicpolypeptide is an artificial signal peptide. In some embodiments, anartificial signal peptide fused to the VZV antigenic polypeptide encodedby the VZV RNA (e.g., mRNA) vaccine is obtained from an immunoglobulinprotein, e.g., an IgE signal peptide or an IgG signal peptide. In someembodiments, a signal peptide fused to the VZV antigenic polypeptideencoded by a VZV RNA (e.g., mRNA) vaccine is an Ig heavy chain epsilon-1signal peptide (IgE HC SP) having the sequence of: MDWTWILFLVAAATRVHS(SEQ ID NO: 56). In some embodiments, a signal peptide fused to a VZVantigenic polypeptide encoded by the VZV RNA (e.g., mRNA) vaccine is anIgGk chain V-III region HAH signal peptide (IgGk SP) having the sequenceof METPAQLLFLLLLWLPDTTG (SEQ ID NO: 57). In some embodiments, the VZVantigenic polypeptide encoded by a VZV RNA (e.g., mRNA) vaccine has anamino acid sequence set forth in one of 10, 14, 18, 22, 26, 30, 34, 38,42 and 45-55 fused to a signal peptide of any one of SEQ ID NO: 56, 57,109, 110 and 111. The examples disclosed herein are not meant to belimiting and any signal peptide that is known in the art to facilitatetargeting of a protein to ER for processing and/or targeting of aprotein to the cell membrane may be used in accordance with the presentdisclosure.

A signal peptide may have a length of 15-60 amino acids. For example, asignal peptide may have a length of 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41,42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,or 60 amino acids. In some embodiments, a signal peptide may have alength of 20-60, 25-60, 30-60, 35-60, 40-60, 45-60, 50-60, 55-60, 15-55,20-55, 25-55, 30-55, 35-55, 40-55, 45-55, 50-55, 15-50, 20-50, 25-50,30-50, 35-50, 40-50, 45-50, 15-45, 20-45, 25-45, 30-45, 35-45, 40-45,15-40, 20-40, 25-40, 30-40, 35-40, 15-35, 20-35, 25-35, 30-35, 15-30,20-30, 25-30, 15-25, 20-25, or 15-20 amino acids.

A signal peptide is typically cleaved from the nascent polypeptide atthe cleavage junction during ER processing. The mature VZV antigenicpolypeptide produce by VZV RNA vaccine of the present disclosuretypically does not comprise a signal peptide.

Chemical Modifications

RNA (e.g., mRNA) vaccines of the present disclosure comprise, in someembodiments, at least one ribonucleic acid (RNA) polynucleotide havingan open reading frame encoding at least one respiratory syncytial virus(VZV) antigenic polypeptide, wherein said RNA comprises at least onechemical modification.

The terms “chemical modification” and “chemically modified” refer tomodification with respect to adenosine (A), guanosine (G), uridine (U),thymidine (T) or cytidine (C) ribonucleosides or deoxyribonucleosides inat least one of their position, pattern, percent or population.Generally, these terms do not refer to the ribonucleotide modificationsin naturally occurring 5′-terminal mRNA cap moieties.

Modifications of polynucleotides include, without limitation, thosedescribed herein, and include, but are expressly not limited to, thosemodifications that comprise chemical modifications. Polynucleotides(e.g., RNA polynucleotides, such as mRNA polynucleotides) may comprisemodifications that are naturally-occurring, non-naturally-occurring orthe polynucleotide may comprise a combination of naturally-occurring andnon-naturally-occurring modifications. Polynucleotides may include anyuseful modification, for example, of a sugar, a nucleobase, or aninternucleoside linkage (e.g., to a linking phosphate, to aphosphodiester linkage or to the phosphodiester backbone).

With respect to a polypeptide, the term “modification” refers to amodification relative to the canonical set of 20 amino acids.Polypeptides, as provided herein, are also considered “modified” if theycontain amino acid substitutions, insertions or a combination ofsubstitutions and insertions.

Polynucleotides (e.g., RNA polynucleotides, such as mRNApolynucleotides), in some embodiments, comprise various (more than one)different modifications. In some embodiments, a particular region of apolynucleotide contains one, two or more (optionally different)nucleoside or nucleotide modifications. In some embodiments, a modifiedRNA polynucleotide (e.g., a modified mRNA polynucleotide), introduced toa cell or organism, exhibits reduced degradation in the cell ororganism, respectively, relative to an unmodified polynucleotide. Insome embodiments, a modified RNA polynucleotide (e.g., a modified mRNApolynucleotide), introduced into a cell or organism, may exhibit reducedimmunogenicity in the cell or organism, respectively (e.g., a reducedinnate response).

Polynucleotides (e.g., RNA polynucleotides, such as mRNApolynucleotides), in some embodiments, comprise non-natural modifiednucleotides that are introduced during synthesis or post-synthesis ofthe polynucleotides to achieve desired functions or properties. Themodifications may be present on internucleotide linkages, purine orpyrimidine bases, or sugars. The modification may be introduced withchemical synthesis or with a polymerase enzyme at the terminal of achain or anywhere else in the chain. Any of the regions of apolynucleotide may be chemically modified.

The present disclosure provides for modified nucleosides and nucleotidesof a polynucleotide (e.g., RNA polynucleotides, such as mRNApolynucleotides). A “nucleoside” refers to a compound containing a sugarmolecule (e.g., a pentose or ribose) or a derivative thereof incombination with an organic base (e.g., a purine or pyrimidine) or aderivative thereof (also referred to herein as “nucleobase”). Anucleotide” refers to a nucleoside, including a phosphate group.Modified nucleotides may by synthesized by any useful method, such as,for example, chemically, enzymatically, or recombinantly, to include oneor more modified or non-natural nucleosides. Polynucleotides maycomprise a region or regions of linked nucleosides. Such regions mayhave variable backbone linkages. The linkages may be standardphosphodiester linkages, in which case the polynucleotides wouldcomprise regions of nucleotides.

Modified nucleotide base pairing encompasses not only the standardadenosine-thymine, adenosine-uracil, or guanosine-cytosine base pairs,but also base pairs formed between nucleotides and/or modifiednucleotides comprising non-standard or modified bases, wherein thearrangement of hydrogen bond donors and hydrogen bond acceptors permitshydrogen bonding between a non-standard base and a standard base orbetween two complementary non-standard base structures, such as, forexample, in those polynucleotides having at least one chemicalmodification. One example of such non-standard base pairing is the basepairing between the modified nucleotide inosine and adenine, cytosine oruracil.

Any combination of base/sugar or linker may be incorporated intopolynucleotides of the present disclosure.

Modifications of polynucleotides (e.g., RNA polynucleotides, such asmRNA polynucleotides), including but not limited to chemicalmodification, that are useful in the compositions, vaccines, methods andsynthetic processes of the present disclosure include, but are notlimited to the following:2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine;2-methylthio-N6-methyladenosine; 2-methylthio-N6-threonylcarbamoyladenosine; N6-glycinylcarbamoyladenosine; N6-isopentenyladenosine; N6-methyladenosine; N6-threonylcarbamoyladenosine;1,2′-O-dimethyladenosine; 1-methyladenosine; 2′-O-methyladenosine;2′-O-ribosyladenosine (phosphate); 2-methyladenosine; 2-methylthio-N6isopentenyladenosine; 2-methylthio-N6-hydroxynorvalylcarbamoyladenosine; 2′-O-methyladenosine; 2′-O-ribosyladenosine(phosphate); Isopentenyladenosine; N6-(cis-hydroxyisopentenyl)adenosine;N6,2′-O-dimethyladenosine; N6,2′-O-dimethyladenosine;N6,N6,2′-O-trimethyladenosine; N6,N6-dimethyladenosine;N6-acetyladenosine; N6-hydroxynorvalylcarbamoyladenosine;N6-methyl-N6-threonylcarbamoyladenosine; 2-methyladenosine;2-methylthio-N6-isopentenyladenosine; 7-deaza-adenosine;N1-methyl-adenosine; N6,N6 (dimethyl)adenine;N6-cis-hydroxy-isopentenyl-adenosine; α-thio-adenosine; 2(amino)adenine; 2 (aminopropyl)adenine; 2 (methylthio) N6(isopentenyl)adenine; 2-(alkyl)adenine; 2-(aminoalkyl)adenine;2-(aminopropyl)adenine; 2-(halo)adenine; 2-(halo)adenine;2-(propyl)adenine; 2′-Amino-2′-deoxy-ATP; 2′-Azido-2′-deoxy-ATP;2′-Deoxy-2′-a-aminoadenosine TP; 2′-Deoxy-2′-a-azidoadenosine TP; 6(alkyl)adenine; 6 (methyl)adenine; 6-(alkyl)adenine; 6-(methyl)adenine;7 (deaza)adenine; 8 (alkenyl)adenine; 8 (alkynyl)adenine; 8(amino)adenine; 8 (thioalkyl)adenine; 8-(alkenyl)adenine;8-(alkyl)adenine; 8-(alkynyl)adenine; 8-(amino)adenine; 8-(halo)adenine;8-(hydroxyl)adenine; 8-(thioalkyl)adenine; 8-(thiol)adenine;8-azido-adenosine; aza adenine; deaza adenine; N6 (methyl)adenine;N6-(isopentyl)adenine; 7-deaza-8-aza-adenosine; 7-methyladenine;1-Deazaadenosine TP; 2′Fluoro-N6-Bz-deoxyadenosine TP;2′-OMe-2-Amino-ATP; 2′O0-methyl-N6-Bz-deoxyadenosine TP;2′-a-Ethynyladenosine TP; 2-aminoadenine; 2-Aminoadenosine TP;2-Amino-ATP; 2′-a-Trifluoromethyladenosine TP; 2-Azidoadenosine TP;2′-b-Ethynyladenosine TP; 2-Bromoadenosine TP;2′-b-Trifluoromethyladenosine TP; 2-Chloroadenosine TP;2′-Deoxy-2′,2′-difluoroadenosine TP; 2′-Deoxy-2′-a-mercaptoadenosine TP;2′-Deoxy-2′-a-thiomethoxyadenosine TP; 2′-Deoxy-2′-b-aminoadenosine TP;2′-Deoxy-2′-b-azidoadenosine TP; 2′-Deoxy-2′-b-bromoadenosine TP;2′-Deoxy-2′-b-chloroadenosine TP; 2′-Deoxy-2′-b-fluoroadenosine TP;2′-Deoxy-2′-b-iodoadenosine TP; 2′-Deoxy-2′-b-mercaptoadenosine TP;2′-Deoxy-2′-b-thiomethoxyadenosine TP; 2-Fluoroadenosine TP;2-Iodoadenosine TP; 2-Mercaptoadenosine TP; 2-methoxy-adenine;2-methylthio-adenine; 2-Trifluoromethyladenosine TP;3-Deaza-3-bromoadenosine TP; 3-Deaza-3-chloroadenosine TP;3-Deaza-3-fluoroadenosine TP; 3-Deaza-3-iodoadenosine TP;3-Deazaadenosine TP; 4′-Azidoadenosine TP; 4′-Carbocyclic adenosine TP;4′-Ethynyladenosine TP; 5′-Homo-adenosine TP; 8-Aza-ATP;8-bromo-adenosine TP; 8-Trifluoromethyladenosine TP; 9-DeazaadenosineTP; 2-aminopurine; 7-deaza-2,6-diaminopurine;7-deaza-8-aza-2,6-diaminopurine; 7-deaza-8-aza-2-aminopurine;2,6-diaminopurine; 7-deaza-8-aza-adenine, 7-deaza-2-aminopurine;2-thiocytidine; 3-methylcytidine; 5-formylcytidine;5-hydroxymethylcytidine; 5-methylcytidine; N4-acetylcytidine;2′-O-methylcytidine; 2′-O-methylcytidine; 5,2′-O-dimethylcytidine;5-formyl-2′-O-methylcytidine; Lysidine; N4,2′-O-dimethylcytidine;N4-acetyl-2′-O-methylcytidine; N4-methylcytidine;N4,N4-Dimethyl-2′-OMe-Cytidine TP; 4-methylcytidine; 5-aza-cytidine;Pseudo-iso-cytidine; pyrrolo-cytidine; α-thio-cytidine;2-(thio)cytosine; 2′-Amino-2′-deoxy-CTP; 2′-Azido-2′-deoxy-CTP;2′-Deoxy-2′-a-aminocytidine TP; 2′-Deoxy-2′-a-azidocytidine TP; 3(deaza) 5 (aza)cytosine; 3 (methyl)cytosine; 3-(alkyl)cytosine;3-(deaza) 5 (aza)cytosine; 3-(methyl)cytidine; 4,2′-O-dimethylcytidine;5 (halo)cytosine; 5 (methyl)cytosine; 5 (propynyl)cytosine; 5(trifluoromethyl)cytosine; 5-(alkyl)cytosine; 5-(alkynyl)cytosine;5-(halo)cytosine; 5-(propynyl)cytosine; 5-(trifluoromethyl)cytosine;5-bromo-cytidine; 5-iodo-cytidine; 5-propynyl cytosine; 6-(azo)cytosine;6-aza-cytidine; aza cytosine; deaza cytosine; N4 (acetyl)cytosine;1-methyl-1-deaza-pseudoisocytidine; 1-methyl-pseudoisocytidine;2-methoxy-5-methyl-cytidine; 2-methoxy-cytidine;2-thio-5-methyl-cytidine; 4-methoxy-1-methyl-pseudoisocytidine;4-methoxy-pseudoisocytidine; 4-thio-1-methyl-1-deaza-pseudoisocytidine;4-thio-1-methyl-pseudoisocytidine; 4-thio-pseudoisocytidine;5-aza-zebularine; 5-methyl-zebularine; pyrrolo-pseudoisocytidine;Zebularine; (E)-5-(2-Bromo-vinyl)cytidine TP; 2,2′-anhydro-cytidine TPhydrochloride; 2′Fluor-N4-Bz-cytidine TP; 2′Fluoro-N4-Acetyl-cytidineTP; 2′-O-Methyl-N4-Acetyl-cytidine TP; 2′O-methyl-N4-Bz-cytidine TP;2′-a-Ethynylcytidine TP; 2′-a-Trifluoromethylcytidine TP;2′-b-Ethynylcytidine TP; 2′-b-Trifluoromethyl cytidine TP;2′-Deoxy-2′,2′-difluorocytidine TP; 2′-Deoxy-2′-a-mercaptocytidine TP;2′-Deoxy-2′-a-thiomethoxycytidine TP; 2′-Deoxy-2′-b-aminocytidine TP;2′-Deoxy-2′-b-azidocytidine TP; 2′-Deoxy-2′-b-bromocytidine TP;2′-Deoxy-2′-b-chlorocytidine TP; 2′-Deoxy-2′-b-fluorocytidine TP;2′-Deoxy-2′-b-iodocytidine TP; 2′-Deoxy-2′-b-mercaptocytidine TP;2′-Deoxy-2′-b-thiomethoxycytidine TP; 2′-O-Methyl-5-(1-propynyl)cytidineTP; 3′-Ethynylcytidine TP; 4′-Azidocytidine TP; 4′-Carbocyclic cytidineTP; 4′-Ethynylcytidine TP; 5-(1-Propynyl)ara-cytidine TP;5-(2-Chloro-phenyl)-2-thiocytidine TP; 5-(4-Amino-phenyl)-2-thiocytidineTP; 5-Aminoallyl-CTP; 5-Cyanocytidine TP; 5-Ethynylara-cytidine TP;5-Ethynylcytidine TP; 5′-Homo-cytidine TP; 5-Methoxycytidine TP;5-Trifluoromethyl-Cytidine TP; N4-Amino-cytidine TP; N4-Benzoyl-cytidineTP; Pseudoisocytidine; 7-methylguanosine; N2,2′-O-dimethylguanosine;N2-methylguanosine; Wyosine; 1,2′-O-dimethylguanosine;1-methylguanosine; 2′-O-methylguanosine; 2′-O-ribosylguanosine(phosphate); 2′-O-methylguanosine; 2′-O-ribosylguanosine (phosphate);7-aminomethyl-7-deazaguanosine; 7-cyano-7-deazaguanosine; Archaeosine;Methylwyosine; N2,7-dimethylguanosine; N2,N2,2′-O-trimethylguanosine;N2,N2,7-trimethylguanosine; N2,N2-dimethylguanosine;N2,7,2′-O-trimethylguanosine; 6-thio-guanosine; 7-deaza-guanosine;8-oxo-guanosine; N1-methyl-guanosine; α-thio-guanosine; 2(propyl)guanine; 2-(alkyl)guanine; 2′-Amino-2′-deoxy-GTP;2′-Azido-2′-deoxy-GTP; 2′-Deoxy-2′-a-aminoguanosine TP;2′-Deoxy-2′-a-azidoguanosine TP; 6 (methyl)guanine; 6-(alkyl)guanine;6-(methyl)guanine; 6-methyl-guanosine; 7 (alkyl)guanine; 7(deaza)guanine; 7 (methyl)guanine; 7-(alkyl)guanine; 7-(deaza)guanine;7-(methyl)guanine; 8 (alkyl)guanine; 8 (alkynyl)guanine; 8(halo)guanine; 8 (thioalkyl)guanine; 8-(alkenyl)guanine;8-(alkyl)guanine; 8-(alkynyl)guanine; 8-(amino)guanine; 8-(halo)guanine;8-(hydroxyl)guanine; 8-(thioalkyl)guanine; 8-(thiol)guanine; azaguanine; deaza guanine; N (methyl)guanine; N-(methyl)guanine;1-methyl-6-thio-guanosine; 6-methoxy-guanosine;6-thio-7-deaza-8-aza-guanosine; 6-thio-7-deaza-guanosine;6-thio-7-methyl-guanosine; 7-deaza-8-aza-guanosine;7-methyl-8-oxo-guanosine; N2,N2-dimethyl-6-thio-guanosine;N2-methyl-6-thio-guanosine; 1-Me-GTP; 2′Fluoro-N2-isobutyl-guanosine TP;2′O-methyl-N2-isobutyl-guanosine TP; 2′-a-Ethynylguanosine TP;2′-a-Trifluoromethylguanosine TP; 2′-b-Ethynylguanosine TP;2′-b-Trifluoromethylguanosine TP; 2′-Deoxy-2′,2′-difluoroguanosine TP;2′-Deoxy-2′-a-mercaptoguanosine TP; 2′-Deoxy-2′-a-thiomethoxyguanosineTP; 2′-Deoxy-2′-b-aminoguanosine TP; 2′-Deoxy-2′-b-azidoguanosine TP;2′-Deoxy-2′-b-bromoguanosine TP; 2′-Deoxy-2′-b-chloroguanosine TP;2′-Deoxy-2′-b-fluoroguanosine TP; 2′-Deoxy-2′-b-iodoguanosine TP;2′-Deoxy-2′-b-mercaptoguanosine TP; 2′-Deoxy-2′-b-thiomethoxyguanosineTP; 4′-Azidoguanosine TP; 4′-Carbocyclic guanosine TP;4′-Ethynylguanosine TP; 5′-Homo-guanosine TP; 8-bromo-guanosine TP;9-Deazaguanosine TP; N2-isobutyl-guanosine TP; 1-methylinosine; Inosine;1,2′-O-dimethylinosine; 2′-O-methylinosine; 7-methylinosine;2′-O-methylinosine; Epoxyqueuosine; galactosyl-queuosine;Mannosylqueuosine; Queuosine; allyamino-thymidine; aza thymidine; deazathymidine; deoxy-thymidine; 2′-O-methyluridine; 2-thiouridine;3-methyluridine; 5-carboxymethyluridine; 5-hydroxyuridine;5-methyluridine; 5-taurinomethyl-2-thiouridine; 5-taurinomethyluridine;Dihydrouridine; Pseudouridine; (3-(3-amino-3-carboxypropyl)uridine;1-methyl-3-(3-amino-5-carboxypropyl)pseudouridine;1-methylpseduouridine; 1-ethyl-pseudouridine; 2′-O-methyluridine;2′-O-methylpseudouridine; 2′-O-methyluridine; 2-thio-2′-O-methyluridine;3-(3-amino-3-carboxypropyl)uridine; 3,2′-O-dimethyluridine;3-Methyl-pseudo-Uridine TP; 4-thiouridine;5-(carboxyhydroxymethyl)uridine; 5-(carboxyhydroxymethyl)uridine methylester; 5,2′-O-dimethyluridine; 5,6-dihydro-uridine;5-aminomethyl-2-thiouridine; 5-carbamoylmethyl-2′-O-methyluridine;5-carbamoylmethyluridine; 5-carboxyhydroxymethyluridine;5-carboxyhydroxymethyluridine methyl ester;5-carboxymethylaminomethyl-2′-O-methyluridine;5-carboxymethylaminomethyl-2-thiouridine;5-carboxymethylaminomethyl-2-thiouridine;5-carboxymethylaminomethyluridine; 5-carboxymethylaminomethyluridine;5-Carbamoylmethyluridine TP; 5-methoxycarbonylmethyl-2′-O-methyluridine;5-methoxycarbonylmethyl-2-thiouridine; 5-methoxycarbonylmethyluridine;5-methyluridine), 5-methoxyuridine; 5-methyl-2-thiouridine; 5-methylaminomethyl-2-selenouridine; 5-methylaminomethyl-2-thiouridine;5-methylaminomethyluridine; 5-Methyldihydrouridine; 5-Oxyaceticacid-Uridine TP; 5-Oxyacetic acid-methyl ester-Uridine TP;N1-methyl-pseudo-uracil; N1-ethyl-pseudo-uracil; uridine 5-oxyaceticacid; uridine 5-oxyacetic acid methyl ester;3-(3-Amino-3-carboxypropyl)-Uridine TP;5-(iso-Pentenylaminomethyl)-2-thiouridine TP;5-(iso-Pentenylaminomethyl)-2′-O-methyluridine TP;5-(iso-Pentenylaminomethyl)uridine TP; 5-propynyl uracil;α-thio-uridine; 1(aminoalkylamino-carbonylethylenyl)-2(thio)-pseudouracil; 1(aminoalkylaminocarbonylethylenyl)-2,4-(dithio)pseudouracil; 1(aminoalkylaminocarbonyl ethylenyl)-4 (thio)pseudouracil; 1(aminoalkylaminocarbonylethylenyl)-pseudouracil; 1(aminocarbonylethylenyl)-2(thio)-pseudouracil; 1(aminocarbonylethylenyl)-2,4-(dithio)pseudouracil; 1 (aminocarbonylethylenyl)-4 (thio)pseudouracil; 1 (aminocarbonyl ethylenyl)-pseudouracil; 1 substituted 2(thio)-pseudouracil; 1 substituted2,4-(dithio)pseudouracil; 1 substituted 4 (thio)pseudouracil; 1substituted pseudouracil;1-(aminoalkylamino-carbonylethylenyl)-2-(thio)-pseudouracil;1-Methyl-3-(3-amino-3-carboxypropyl) pseudouridine TP;1-Methyl-3-(3-amino-3-carboxypropyl)pseudo-UTP; 1-Methyl-pseudo-UTP;1-Ethyl-pseudo-UTP; 2 (thio)pseudouracil; 2′ deoxy uridine; 2′fluorouridine; 2-(thio)uracil; 2,4-(dithio)psuedouracil; 2′ methyl,2′amino, 2′azido, 2′fluro-guanosine; 2′-Amino-2′-deoxy-UTP;2′-Azido-2′-deoxy-UTP; 2′-Azido-deoxyuridine TP;2′-O-methylpseudouridine; 2′ deoxy uridine; 2′ fluorouridine;2′-Deoxy-2′-a-aminouridine TP; 2′-Deoxy-2′-a-azidouridine TP;2-methylpseudouridine; 3 (3 amino-3 carboxypropyl)uracil; 4(thio)pseudouracil; 4-(thio)pseudouracil; 4-(thio)uracil; 4-thiouracil;5 (1,3-diazole-1-alkyl)uracil; 5 (2-aminopropyl)uracil; 5(aminoalkyl)uracil; 5 (dimethylaminoalkyl)uracil; 5(guanidiniumalkyl)uracil; 5 (methoxycarbonylmethyl)-2-(thio)uracil; 5(methoxycarbonyl-methyl)uracil; 5 (methyl) 2 (thio)uracil; 5 (methyl)2,4 (dithio)uracil; 5 (methyl) 4 (thio)uracil; 5 (methylaminomethyl)-2(thio)uracil; 5 (methylaminomethyl)-2,4 (dithio)uracil; 5(methylaminomethyl)-4 (thio)uracil; 5 (propynyl)uracil; 5(trifluoromethyl)uracil; 5-(2-aminopropyl)uracil;5-(alkyl)-2-(thio)pseudouracil; 5-(alkyl)-2,4 (dithio)pseudouracil;5-(alkyl)-4 (thio)pseudouracil; 5-(alkyl)pseudouracil; 5-(alkyl)uracil;5-(alkynyl)uracil; 5-(allylamino)uracil; 5-(cyanoalkyl)uracil;5-(dialkylaminoalkyl)uracil; 5-(dimethylaminoalkyl)uracil;5-(guanidiniumalkyl)uracil; 5-(halo)uracil;5-(1,3-diazole-1-alkyl)uracil; 5-(methoxy)uracil;5-(methoxycarbonylmethyl)-2-(thio)uracil;5-(methoxycarbonyl-methyl)uracil; 5-(methyl) 2(thio)uracil; 5-(methyl)2,4 (dithio)uracil; 5-(methyl) 4 (thio)uracil;5-(methyl)-2-(thio)pseudouracil; 5-(methyl)-2,4 (dithio)pseudouracil;5-(methyl)-4 (thio)pseudouracil; 5-(methyl)pseudouracil;5-(methylaminomethyl)-2 (thio)uracil;5-(methylaminomethyl)-2,4(dithio)uracil;5-(methylaminomethyl)-4-(thio)uracil; 5-(propynyl)uracil;5-(trifluoromethyl)uracil; 5-aminoallyl-uridine; 5-bromo-uridine;5-iodo-uridine; 5-uracil; 6 (azo)uracil; 6-(azo)uracil; 6-aza-uridine;allyamino-uracil; aza uracil; deaza uracil; N3 (methyl)uracil; Pseudo-UTP-1-2-ethanoic acid; Pseudouracil; 4-Thio-pseudo-UTP;1-carboxymethyl-pseudouridine; 1-methyl-1-deaza-pseudouridine;1-propynyl-uridine; 1-taurinomethyl-1-methyl-uridine;1-taurinomethyl-4-thio-uridine; 1-taurinomethyl-pseudouridine;2-methoxy-4-thio-pseudouridine; 2-thio-1-methyl-1-deaza-pseudouridine;2-thio-1-methyl-pseudouridine; 2-thio-5-aza-uridine;2-thio-dihydropseudouridine; 2-thio-dihydrouridine;2-thio-pseudouridine; 4-methoxy-2-thio-pseudouridine;4-methoxy-pseudouridine; 4-thio-1-methyl-pseudouridine;4-thio-pseudouridine; 5-aza-uridine; Dihydropseudouridine;(±)1-(2-Hydroxypropyl)pseudouridine TP;(2R)-1-(2-Hydroxypropyl)pseudouridine TP;(2S)-1-(2-Hydroxypropyl)pseudouridine TP;(E)-5-(2-Bromo-vinyl)ara-uridine TP; (E)-5-(2-Bromo-vinyl)uridine TP;(Z)-5-(2-Bromo-vinyl)ara-uridine TP; (Z)-5-(2-Bromo-vinyl)uridine TP;1-(2,2,2-Trifluoroethyl)-pseudo-UTP;1-(2,2,3,3,3-Pentafluoropropyl)pseudouridine TP;1-(2,2-Diethoxyethyl)pseudouridine TP;1-(2,4,6-Trimethylbenzyl)pseudouridine TP;1-(2,4,6-Trimethyl-benzyl)pseudo-UTP;1-(2,4,6-Trimethyl-phenyl)pseudo-UTP;1-(2-Amino-2-carboxyethyl)pseudo-UTP; 1-(2-Amino-ethyl)pseudo-UTP;1-(2-Hydroxyethyl)pseudouridine TP; 1-(2-Methoxyethyl)pseudouridine TP;1-(3,4-Bis-trifluoromethoxybenzyl)pseudouridine TP;1-(3,4-Dimethoxybenzyl)pseudouridine TP;1-(3-Amino-3-carboxypropyl)pseudo-UTP; 1-(3-Amino-propyl)pseudo-UTP;1-(3-Cyclopropyl-prop-2-ynyl)pseudouridine TP;1-(4-Amino-4-carboxybutyl)pseudo-UTP; 1-(4-Amino-benzyl)pseudo-UTP;1-(4-Amino-butyl)pseudo-UTP; 1-(4-Amino-phenyl)pseudo-UTP;1-(4-Azidobenzyl)pseudouridine TP; 1-(4-Bromobenzyl)pseudouridine TP;1-(4-Chlorobenzyl)pseudouridine TP; 1-(4-Fluorobenzyl)pseudouridine TP;1-(4-Iodobenzyl)pseudouridine TP;1-(4-Methanesulfonylbenzyl)pseudouridine TP;1-(4-Methoxybenzyl)pseudouridine TP; 1-(4-Methoxy-benzyl)pseudo-UTP;1-(4-Methoxy-phenyl)pseudo-UTP; 1-(4-Methylbenzyl)pseudouridine TP;1-(4-Methyl-benzyl)pseudo-UTP; 1-(4-Nitrobenzyl)pseudouridine TP;1-(4-Nitro-benzyl)pseudo-UTP; 1(4-Nitro-phenyl)pseudo-UTP;1-(4-Thiomethoxybenzyl)pseudouridine TP;1-(4-Trifluoromethoxybenzyl)pseudouridine TP;1-(4-Trifluoromethylbenzyl)pseudouridine TP;1-(5-Amino-pentyl)pseudo-UTP; 1-(6-Amino-hexyl)pseudo-UTP;1,6-Dimethyl-pseudo-UTP;1-[3-(2-{2-[2-(2-Aminoethoxy)-ethoxy]-ethoxy}-ethoxy)-propionyl]pseudouridineTP; 1-{3-[2-(2-Aminoethoxy)-ethoxy]-propionyl}pseudouridine TP;1-Acetylpseudouridine TP; 1-Alkyl-6-(1-propynyl)-pseudo-UTP;1-Alkyl-6-(2-propynyl)-pseudo-UTP; 1-Alkyl-6-allyl-pseudo-UTP;1-Alkyl-6-ethynyl-pseudo-UTP; 1-Alkyl-6-homoallyl-pseudo-UTP;1-Alkyl-6-vinyl-pseudo-UTP; 1-Allylpseudouridine TP;1-Aminomethyl-pseudo-UTP; 1-Benzoylpseudouridine TP;1-Benzyloxymethylpseudouridine TP; 1-Benzyl-pseudo-UTP;1-Biotinyl-PEG2-pseudouridine TP; 1-Biotinylpseudouridine TP;1-Butyl-pseudo-UTP; 1-Cyanomethylpseudouridine TP;1-Cyclobutylmethyl-pseudo-UTP; 1-Cyclobutyl-pseudo-UTP;1-Cycloheptylmethyl-pseudo-UTP; 1-Cycloheptyl-pseudo-UTP;1-Cyclohexylmethyl-pseudo-UTP; 1-Cyclohexyl-pseudo-UTP;1-Cyclooctylmethyl-pseudo-UTP; 1-Cyclooctyl-pseudo-UTP;1-Cyclopentylmethyl-pseudo-UTP; 1-Cyclopentyl-pseudo-UTP;1-Cyclopropylmethyl-pseudo-UTP; 1-Cyclopropyl-pseudo-UTP;1-Ethyl-pseudo-UTP; 1-Hexyl-pseudo-UTP; 1-Homoallylpseudouridine TP;1-Hydroxymethylpseudouridine TP; 1-iso-propyl-pseudo-UTP;1-Me-2-thio-pseudo-UTP; 1-Me-4-thio-pseudo-UTP;1-Me-alpha-thio-pseudo-UTP; 1-Methanesulfonylmethylpseudouridine TP;1-Methoxymethylpseudouridine TP;1-Methyl-6-(2,2,2-Trifluoroethyl)pseudo-UTP;1-Methyl-6-(4-morpholino)-pseudo-UTP;1-Methyl-6-(4-thiomorpholino)-pseudo-UTP; 1-Methyl-6-(substitutedphenyl)pseudo-UTP; 1-Methyl-6-amino-pseudo-UTP;1-Methyl-6-azido-pseudo-UTP; 1-Methyl-6-bromo-pseudo-UTP;1-Methyl-6-butyl-pseudo-UTP; 1-Methyl-6-chloro-pseudo-UTP;1-Methyl-6-cyano-pseudo-UTP; 1-Methyl-6-dimethylamino-pseudo-UTP;1-Methyl-6-ethoxy-pseudo-UTP; 1-Methyl-6-ethylcarboxylate-pseudo-UTP;1-Methyl-6-ethyl-pseudo-UTP; 1-Methyl-6-fluoro-pseudo-UTP;1-Methyl-6-formyl-pseudo-UTP; 1-Methyl-6-hydroxyamino-pseudo-UTP;1-Methyl-6-hydroxy-pseudo-UTP; 1-Methyl-6-iodo-pseudo-UTP;1-Methyl-6-iso-propyl-pseudo-UTP; 1-Methyl-6-methoxy-pseudo-UTP;1-Methyl-6-methylamino-pseudo-UTP; 1-Methyl-6-phenyl-pseudo-UTP;1-Methyl-6-propyl-pseudo-UTP; 1-Methyl-6-tert-butyl-pseudo-UTP;1-Methyl-6-trifluoromethoxy-pseudo-UTP;1-Methyl-6-trifluoromethyl-pseudo-UTP; 1-MorpholinomethylpseudouridineTP; 1-Pentyl-pseudo-UTP; 1-Phenyl-pseudo-UTP; 1-PivaloylpseudouridineTP; 1-Propargylpseudouridine TP; 1-Propyl-pseudo-UTP;1-propynyl-pseudouridine; 1-p-tolyl-pseudo-UTP; 1-tert-Butyl-pseudo-UTP;1-Thiomethoxymethylpseudouridine TP; 1-ThiomorpholinomethylpseudouridineTP; 1-Trifluoroacetylpseudouridine TP; 1-Trifluoromethyl-pseudo-UTP;1-Vinylpseudouridine TP; 2,2′-anhydro-uridine TP; 2′-bromo-deoxyuridineTP; 2′-F-5-Methyl-2′-deoxy-UTP; 2′-OMe-5-Me-UTP; 2′-OMe-pseudo-UTP;2′-a-Ethynyluridine TP; 2′-a-Trifluoromethyluridine TP;2′-b-Ethynyluridine TP; 2′-b-Trifluoromethyluridine TP;2′-Deoxy-2′,2′-difluorouridine TP; 2′-Deoxy-2′-a-mercaptouridine TP;2′-Deoxy-2′-a-thiomethoxyuridine TP; 2′-Deoxy-2′-b-aminouridine TP;2′-Deoxy-2′-b-azidouridine TP; 2′-Deoxy-2′-b-bromouridine TP;2′-Deoxy-2′-b-chlorouridine TP; 2′-Deoxy-2′-b-fluorouridine TP;2′-Deoxy-2′-b-iodouridine TP; 2′-Deoxy-2′-b-mercaptouridine TP;2′-Deoxy-2′-b-thiomethoxyuridine TP; 2-methoxy-4-thio-uridine;2-methoxyuridine; 2′-O-Methyl-5-(1-propynyl)uridine TP;3-Alkyl-pseudo-UTP; 4′-Azidouridine TP; 4′-Carbocyclic uridine TP;4′-Ethynyluridine TP; 5-(1-Propynyl)ara-uridine TP; 5-(2-Furanyl)uridineTP; 5-Cyanouridine TP; 5-Dimethyl aminouridine TP; 5′-Homo-uridine TP;5-iodo-2′-fluoro-deoxyuridine TP; 5-Phenylethynyluridine TP;5-Trideuteromethyl-6-deuterouridine TP; 5-Trifluoromethyl-Uridine TP;5-Vinylarauridine TP; 6-(2,2,2-Trifluoroethyl)-pseudo-UTP;6-(4-Morpholino)-pseudo-UTP; 6-(4-Thiomorpholino)-pseudo-UTP;6-(Substituted-Phenyl)-pseudo-UTP; 6-Amino-pseudo-UTP;6-Azido-pseudo-UTP; 6-Bromo-pseudo-UTP; 6-Butyl-pseudo-UTP;6-Chloro-pseudo-UTP; 6-Cyano-pseudo-UTP; 6-Dimethylamino-pseudo-UTP;6-Ethoxy-pseudo-UTP; 6-Ethylcarboxylate-pseudo-UTP; 6-Ethyl-pseudo-UTP;6-Fluoro-pseudo-UTP; 6-Formyl-pseudo-UTP; 6-Hydroxyamino-pseudo-UTP;6-Hydroxy-pseudo-UTP; 6-Iodo-pseudo-UTP; 6-iso-Propyl-pseudo-UTP;6-Methoxy-pseudo-UTP; 6-Methylamino-pseudo-UTP; 6-Methyl-pseudo-UTP;6-Phenyl-pseudo-UTP; 6-Phenyl-pseudo-UTP; 6-Propyl-pseudo-UTP;6-tert-Butyl-pseudo-UTP; 6-Trifluoromethoxy-pseudo-UTP;6-Trifluoromethyl-pseudo-UTP; Alpha-thio-pseudo-UTP; Pseudouridine1-(4-methylbenzenesulfonic acid) TP; Pseudouridine 1-(4-methylbenzoicacid) TP; Pseudouridine TP 1-[3-(2-ethoxy)]propionic acid; PseudouridineTP 1-[3-{2-(2-[2-(2-ethoxy)-ethoxy]-ethoxy)-ethoxy}]propionic acid;Pseudouridine TP1-[3-{2-(2-[2-{2(2-ethoxy)-ethoxy})-ethoxy]-ethoxy)-ethoxy}]propionicacid; Pseudouridine TP 1-[3-{2-(2-[2-ethoxy]-ethoxy)-ethoxy}]propionicacid; Pseudouridine TP 1-[3-{2-(2-ethoxy)-ethoxy}] propionic acid;Pseudouridine TP 1-methylphosphonic acid; Pseudouridine TP1-methylphosphonic acid diethyl ester; Pseudo-UTP-N1-3-propionic acid;Pseudo-UTP-N1-4-butanoic acid; Pseudo-UTP-N1-5-pentanoic acid;Pseudo-UTP-N1-6-hexanoic acid; Pseudo-UTP-N1-7-heptanoic acid;Pseudo-UTP-N1-methyl-p-benzoic acid; Pseudo-UTP-N1-p-benzoic acid;Wybutosine; Hydroxywybutosine; Isowyosine; Peroxywybutosine;undermodified hydroxywybutosine; 4-demethylwyosine; 2,6-(diamino)purine;1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl:1,3-(diaza)-2-(oxo)-phenthiazin-1-yl;1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;1,3,5-(triaza)-2,6-(dioxa)-naphthalene; 2 (amino)purine;2,4,5-(trimethyl)phenyl; 2′ methyl, 2′amino, 2′azido, 2′fluro-cytidine;2′ methyl, 2′amino, 2′azido, 2′fluro-adenine; 2′methyl, 2′amino,2′azido, 2′fluro-uridine; 2′-amino-2′-deoxyribose;2-amino-6-Chloro-purine; 2-aza-inosinyl; 2′-azido-2′-deoxyribose;2′fluoro-2′-deoxyribose; 2′-fluoro-modified bases; 2′-O-methyl-ribose;2-oxo-7-aminopyridopyrimidin-3-yl; 2-oxo-pyridopyrimidine-3-yl;2-pyridinone; 3 nitropyrrole; 3-(methyl)-7-(propynyl)isocarbostyrilyl;3-(methyl)isocarbostyrilyl; 4-(fluoro)-6-(methyl)benzimidazole;4-(methyl)benzimidazole; 4-(methyl)indolyl; 4,6-(dimethyl)indolyl; 5nitroindole; 5 substituted pyrimidines; 5-(methyl)isocarbostyrilyl;5-nitroindole; 6-(aza)pyrimidine; 6-(azo)thymine;6-(methyl)-7-(aza)indolyl; 6-chloro-purine;6-phenyl-pyrrolo-pyrimidin-2-on-3-yl;7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl;7-(aminoalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl;7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl;7-(aminoalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;7-(aza)indolyl;7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazinl-yl;7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenthiazin-1-yl;7-(guanidiniumalkylhydroxy)-1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl;7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;7-(guanidiniumalkyl-hydroxy)-1,3-(diaza)-2-(oxo)-phenthiazin-1-yl;7-(guanidiniumalkylhydroxy)-1,3-(diaza)-2-(oxo)-phenoxazin-1-yl;7-(propynyl)isocarbostyrilyl; 7-(propynyl)isocarbostyrilyl,propynyl-7-(aza)indolyl; 7-deaza-inosinyl; 7-substituted1-(aza)-2-(thio)-3-(aza)-phenoxazin-1-yl; 7-substituted1,3-(diaza)-2-(oxo)-phenoxazin-1-yl; 9-(methyl)-imidizopyridinyl;Aminoindolyl; Anthracenyl;bis-ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl;bis-ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl;Difluorotolyl; Hypoxanthine; Imidizopyridinyl; Inosinyl;Isocarbostyrilyl; Isoguanisine; N2-substituted purines;N6-methyl-2-amino-purine; N6-substituted purines; N-alkylatedderivative; Napthalenyl; Nitrobenzimidazolyl; Nitroimidazolyl;Nitroindazolyl; Nitropyrazolyl; Nubularine; O6-substituted purines;O-alkylated derivative;ortho-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl;ortho-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; Oxoformycin TP;para-(aminoalkylhydroxy)-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl;para-substituted-6-phenyl-pyrrolo-pyrimidin-2-on-3-yl; Pentacenyl;Phenanthracenyl; Phenyl; propynyl-7-(aza)indolyl; Pyrenyl;pyridopyrimidin-3-yl; pyridopyrimidin-3-yl,2-oxo-7-amino-pyridopyrimidin-3-yl; pyrrolo-pyrimidin-2-on-3-yl;Pyrrolopyrimidinyl; Pyrrolopyrizinyl; Stilbenzyl; substituted1,2,4-triazoles; Tetracenyl; Tubercidine; Xanthine; Xanthosine-5′-TP;2-thio-zebularine; 5-aza-2-thio-zebularine; 7-deaza-2-amino-purine;pyridin-4-one ribonucleoside; 2-Amino-riboside-TP; Formycin A TP;Formycin B TP; Pyrrolosine TP; 2′-OH-ara-adenosine TP;2′-OH-ara-cytidine TP; 2′-OH-ara-uridine TP; 2′-OH-ara-guanosine TP;5-(2-carbomethoxyvinyl)uridine TP; andN6-(19-Amino-pentaoxanonadecyl)adenosine TP.

In some embodiments, polynucleotides (e.g., RNA polynucleotides, such asmRNA polynucleotides) include a combination of at least two (e.g., 2, 3,4 or more) of the aforementioned modified nucleobases.

In some embodiments, modified nucleobases in polynucleotides (e.g., RNApolynucleotides, such as mRNA polynucleotides) are selected from thegroup consisting of pseudouridine (ψ), 2-thiouridine (s2U),4′-thiouridine, 5-methylcytosine, 2-thio-1-methyl-1-deaza-pseudouridine,2-thio-1-methyl-pseudouridine, 2-thio-5-aza-uridine,2-thio-dihydropseudouridine, 2-thio-dihydrouridine,2-thio-pseudouridine, 4-methoxy-2-thio-pseudouridine,4-methoxy-pseudouridine, 4-thio-1-methyl-pseudouridine,4-thio-pseudouridine, 5-aza-uridine, dihydropseudouridine,5-methyluridine, 5-methoxyuridine, 2′-O-methyl uridine,1-methyl-pseudouridine (m1ψ), 1-ethyl-pseudouridine (e1ψ),5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), α-thio-guanosine,α-thio-adenosine, 5-cyano uridine, 4′-thio uridine 7-deaza-adenine,1-methyl-adenosine (m1A), 2-methyl-adenine (m2A), N6-methyl-adenosine(m6A), and 2,6-Diaminopurine, (I), 1-methyl-inosine (m1I), wyosine(imG), methylwyosine (mimG), 7-deaza-guanosine,7-cyano-7-deaza-guanosine (preQ0), 7-aminomethyl-7-deaza-guanosine(preQ1), 7-methyl-guanosine (m7G), 1-methyl-guanosine (ml 1G),8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 2,8-dimethyl adenosine,2-geranylthiouridine, 2-lysidine, 2-selenouridine,3-(3-amino-3-carboxypropyl)-5,6-dihydrouridine,3-(3-amino-3-carboxypropyl)pseudouridine, 3-methylpseudouridine,5-(carboxyhydroxymethyl)-2′-O-methyluridine methyl ester,5-aminomethyl-2-geranylthiouridine, 5-aminomethyl-2-selenouridine,5-aminomethyluridine, 5-carbamoylhydroxymethyluridine,5-carbamoylmethyl-2-thiouridine, 5-carboxymethyl-2-thiouridine,5-carboxymethylaminomethyl-2-geranylthiouridine,5-carboxymethylaminomethyl-2-selenouridine, 5-cyanomethyluridine,5-hydroxycytidine, 5-methylaminomethyl-2-geranylthiouridine,7-aminocarboxypropyl-demethylwyosine, 7-aminocarboxypropylwyosine,7-aminocarboxypropylwyosine methyl ester, 8-methyladenosine,N4,N4-dimethyl cytidine, N6-formyladenosine, N6-hydroxymethyladenosine,agmatidine, cyclic N6-threonylcarbamoyladenosine, glutamyl-queuosine,methylated undermodified hydroxywybutosine,N4,N4,2′-O-trimethylcytidine, geranylated5-methylaminomethyl-2-thiouridine, geranylated5-carboxymethylaminomethyl-2-thiouridine, Qbase, preQ0base, preQ1base,and combinations of two or more thereof. In some embodiments, the atleast one chemically modified nucleoside is selected from the groupconsisting of pseudouridine, 1-methyl-pseudouridine,1-ethyl-pseudouridine, 5-methylcytosine, 5-methoxyuridine, and acombination thereof. In some embodiments, the polyribonucleotide (e.g.,RNA polyribonucleotide, such as mRNA polyribonucleotide) includes acombination of at least two (e.g., 2, 3, 4 or more) of theaforementioned modified nucleobases. In some embodiments,polynucleotides (e.g., RNA polynucleotides, such as mRNApolynucleotides) include a combination of at least two (e.g., 2, 3, 4 ormore) of the aforementioned modified nucleobases.

In some embodiments, modified nucleobases in polynucleotides (e.g., RNApolynucleotides, such as mRNA polynucleotides) are selected from thegroup consisting of 1-methyl-pseudouridine (m1ψ), 1-ethyl-pseudouridine(e1ψ), 5-methoxy-uridine (mo5U), 5-methyl-cytidine (m5C), pseudouridine(ψ), α-thio-guanosine and α-thio-adenosine. In some embodiments, thepolyribonucleotide includes a combination of at least two (e.g., 2, 3, 4or more) of the aforementioned modified nucleobases, including but notlimited to chemical modifications.

In some embodiments, polynucleotides (e.g., RNA polynucleotides, such asmRNA polynucleotides) comprise pseudouridine (ψ) and 5-methyl-cytidine(m5C). In some embodiments, the polyribonucleotides (e.g., RNA, such asmRNA) comprise 1-methyl-pseudouridine (m1ψ). In some embodiments, thepolyribonucleotides (e.g., RNA, such as mRNA) comprise1-ethyl-pseudouridine (e1ψ). In some embodiments, thepolyribonucleotides (e.g., RNA, such as mRNA) comprise1-methyl-pseudouridine (m1ψ) and 5-methyl-cytidine (m5C). In someembodiments, the polyribonucleotides (e.g., RNA, such as mRNA) comprise1-ethyl-pseudouridine (e1ψ) and 5-methyl-cytidine (m5C). In someembodiments, the polyribonucleotides (e.g., RNA, such as mRNA) comprise2-thiouridine (s2U). In some embodiments, the polyribonucleotides (e.g.,RNA, such as mRNA) comprise 2-thiouridine and 5-methyl-cytidine (m5C).In some embodiments, the polyribonucleotides (e.g., RNA, such as mRNA)comprise methoxy-uridine (mo5U). In some embodiments, thepolyribonucleotides (e.g., RNA, such as mRNA) comprise 5-methoxy-uridine(mo5U) and 5-methyl-cytidine (m5C). In some embodiments, thepolyribonucleotides (e.g., RNA, such as mRNA) comprise 2′-O-methyluridine. In some embodiments, the polyribonucleotides (e.g., RNA, suchas mRNA) comprise 2′-O-methyl uridine and 5-methyl-cytidine (m5C). Insome embodiments, the polyribonucleotides (e.g., RNA, such as mRNA)comprise N6-methyl-adenosine (m6A). In some embodiments, thepolyribonucleotides (e.g., RNA, such as mRNA) compriseN6-methyl-adenosine (m6A) and 5-methyl-cytidine (m5C).

In some embodiments, polynucleotides (e.g., RNA polynucleotides, such asmRNA polynucleotides) are uniformly modified (e.g., fully modified,modified throughout the entire sequence) for a particular modification.For example, a polynucleotide can be uniformly modified with1-methyl-pseudouridine, meaning that all uridine residues in the mRNAsequence are replaced with 1-methyl-pseudouridine. Similarly, apolynucleotide can be uniformly modified for any type of nucleosideresidue present in the sequence by replacement with a modified residuesuch as those set forth above.

Exemplary nucleobases and nucleosides having a modified cytosine includeN4-acetyl-cytidine (ac4C), 5-methyl-cytidine (m5C), 5-halo-cytidine(e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm5C),1-methyl-pseudoisocytidine, 2-thio-cytidine (s2C), and2-thio-5-methyl-cytidine.

In some embodiments, a modified nucleobase is a modified uridine.Exemplary nucleobases and nucleosides having a modified uridine include1-methyl-pseudouridine (m1ψ), 1-ethyl-pseudouridine (e1ψ), 5-methoxyuridine, 2-thio uridine, 5-cyano uridine, 2′-O-methyl uridine and4′-thio uridine.

In some embodiments, a modified nucleobase is a modified adenine.Exemplary nucleobases and nucleosides having a modified adenine include7-deaza-adenine, 1-methyl-adenosine (m1A), 2-methyl-adenine (m2A), andN6-methyl-adenosine (m6A).

In some embodiments, a modified nucleobase is a modified guanine.Exemplary nucleobases and nucleosides having a modified guanine includeinosine (I), 1-methyl-inosine (m1I), wyosine (imG), methylwyosine(mimG), 7-deaza-guanosine, 7-cyano-7-deaza-guanosine (preQ0),7-aminomethyl-7-deaza-guanosine (preQ1), 7-methyl-guanosine (m7G),1-methyl-guanosine (m1G), 8-oxo-guanosine, and 7-methyl-8-oxo-guanosine.

The polynucleotides of the present disclosure may be partially or fullymodified along the entire length of the molecule. For example, one ormore or all or a given type of nucleotide (e.g., purine or pyrimidine,or any one or more or all of A, G, U, C) may be uniformly modified in apolynucleotide of the invention, or in a given predetermined sequenceregion thereof (e.g., in the mRNA including or excluding the polyAtail). In some embodiments, all nucleotides X in a polynucleotide of thepresent disclosure (or in a given sequence region thereof) are modifiednucleotides, wherein X may be any one of nucleotides A, G, U, C, or anyone of the combinations A+G, A+U, A+C, G+U, G+C, U+C, A+G+U, A+G+C,G+U+C or A+G+C.

The polynucleotide may contain from about 1% to about 100% modifiednucleotides (either in relation to overall nucleotide content, or inrelation to one or more types of nucleotide, i.e., any one or more of A,G, U or C) or any intervening percentage (e.g., from 1% to 20%, from 1%to 25%, from 1% to 50%, from 1% to 60%, from 1% to 70%, from 1% to 80%,from 1% to 90%, from 1% to 95%, from 10% to 20%, from 10% to 25%, from10% to 50%, from 10% to 60%, from 10% to 70%, from 10% to 80%, from 10%to 90%, from 10% to 95%, from 10% to 100%, from 20% to 25%, from 20% to50%, from 20% to 60%, from 20% to 70%, from 20% to 80%, from 20% to 90%,from 20% to 95%, from 20% to 100%, from 50% to 60%, from 50% to 70%,from 50% to 80%, from 50% to 90%, from 50% to 95%, from 50% to 100%,from 70% to 80%, from 70% to 90%, from 70% to 95%, from 70% to 100%,from 80% to 90%, from 80% to 95%, from 80% to 100%, from 90% to 95%,from 90% to 100%, and from 95% to 100%). It will be understood that anyremaining percentage is accounted for by the presence of unmodified A,G, U, or C.

The polynucleotides may contain at a minimum 1% and at maximum 100%modified nucleotides, or any intervening percentage, such as at least 5%modified nucleotides, at least 10% modified nucleotides, at least 25%modified nucleotides, at least 50% modified nucleotides, at least 80%modified nucleotides, or at least 90% modified nucleotides. For example,the polynucleotides may contain a modified pyrimidine such as a modifieduracil or cytosine. In some embodiments, at least 5%, at least 10%, atleast 25%, at least 50%, at least 80%, at least 90% or 100% of theuracil in the polynucleotide is replaced with a modified uracil (e.g., a5-substituted uracil). The modified uracil can be replaced by a compoundhaving a single unique structure, or can be replaced by a plurality ofcompounds having different structures (e.g., 2, 3, 4 or more uniquestructures). In some embodiments, at least 5%, at least 10%, at least25%, at least 50%, at least 80%, at least 90% or 100% of the cytosine inthe polynucleotide is replaced with a modified cytosine (e.g., a5-substituted cytosine). The modified cytosine can be replaced by acompound having a single unique structure, or can be replaced by aplurality of compounds having different structures (e.g., 2, 3, 4 ormore unique structures).

Thus, in some embodiments, the RNA vaccines comprise a 5′UTR element, anoptionally codon optimized open reading frame, and a 3′UTR element, apoly(A) sequence and/or a polyadenylation signal wherein the RNA is notchemically modified.

In some embodiments, the modified nucleobase is a modified uracil.Exemplary nucleobases and nucleosides having a modified uracil includepseudouridine (ψ), pyridin-4-one ribonucleoside, 5-aza-uridine,6-aza-uridine, 2-thio-5-aza-uridine, 2-thio-uridine (s²U),4-thio-uridine (s⁴U), 4-thio-pseudouridine, 2-thio-pseudouridine,5-hydroxy-uridine (ho⁵U), 5-aminoallyl-uridine, 5-halo-uridine (e.g.,5-iodo-uridineor 5-bromo-uridine), 3-methyl-uridine (m³U),5-methoxy-uridine (mo⁵U), uridine 5-oxyacetic acid (cmo⁵U), uridine5-oxyacetic acid methyl ester (mcmo⁵U), 5-carboxymethyl-uridine (cm⁵U),1-carboxymethyl-pseudouridine, 5-carboxyhydroxymethyl-uridine (chm⁵U),5-carboxyhydroxymethyl-uridine methyl ester (mchm⁵U),5-methoxycarbonylmethyl-uridine (mcm⁵U),5-methoxycarbonylmethyl-2-thio-uridine (mcm⁵ s²U),5-aminomethyl-2-thio-uridine (nm⁵ s²U), 5-methylaminomethyl-uridine(mnm⁵U), 5-methylaminomethyl-2-thio-uridine (mnm⁵s²U),5-methylaminomethyl-2-seleno-uridine (mnm⁵se²U),5-carbamoylmethyl-uridine (ncm⁵U), 5-carboxymethylaminomethyl-uridine(cmnm⁵U), 5-carboxymethylaminomethyl-2-thio-uridine (cmnm⁵s²U),5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyl-uridine(τm⁵U), 1-taurinomethyl-pseudouridine, 5-taurinomethyl-2-thio-uridine(τm⁵ s²U), 1-taurinomethyl-4-thio-pseudouridine, 5-methyl-uridine (m⁵U,i.e., having the nucleobase deoxythymine), 1-methyl-pseudouridine (m¹ψ),1-ethyl-pseudouridine (e1ψ), 5-methyl-2-thio-uridine (m⁵s²U),1-methyl-4-thio-pseudouridine (m¹s⁴ψ), 4-thio-1-methyl-pseudouridine,3-methyl-pseudouridine (m³ψ), 2-thio-1-methyl-pseudouridine,1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-1-deaza-pseudouridine,dihydrouridine (D), dihydropseudouridine, 5,6-dihydrouridine,5-methyl-dihydrouridine (m⁵D), 2-thio-dihydrouridine,2-thio-dihydropseudouridine, 2-methoxy-uridine,2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine,4-methoxy-2-thio-pseudouridine, N1-methyl-pseudouridine,3-(3-amino-3-carboxypropyl)uridine (acp³U),1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine (acp³ ψ),5-(isopentenylaminomethyl)uridine (inm⁵U),5-(isopentenylaminomethyl)-2-thio-uridine (inm⁵s²U), α-thio-uridine,2′-O-methyl-uridine (Um), 5,2′-O-dimethyl-uridine (m⁵Um),2′-O-methyl-pseudouridine (ψm), 2-thio-2′-O-methyl-uridine (s²Um),5-methoxycarbonylmethyl-2′-O-methyl-uridine (mcm⁵Um),5-carbamoylmethyl-2′-O-methyl-uridine (ncm⁵Um),5-carboxymethylaminomethyl-2′-O-methyl-uridine (cmnm⁵Um),3,2′-O-dimethyl-uridine (m³Um), and5-(isopentenylaminomethyl)-2′-O-methyl-uridine (inm⁵Um), 1-thio-uridine,deoxythymidine, 2′-F-ara-uridine, 2′-F-uridine, 2′-OH-ara-uridine,5-(2-carbomethoxyvinyl) uridine, and 5-[3-(1-E-propenylamino)]uridine.

In some embodiments, the modified nucleobase is a modified cytosine.Exemplary nucleobases and nucleosides having a modified cytosine include5-aza-cytidine, 6-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine(m³C), N4-acetyl-cytidine (ac⁴C), 5-formyl-cytidine (f⁵C),N4-methyl-cytidine (m⁴C), 5-methyl-cytidine (m⁵C), 5-halo-cytidine(e.g., 5-iodo-cytidine), 5-hydroxymethyl-cytidine (hm⁵C),1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine,2-thio-cytidine (s²C), 2-thio-5-methyl-cytidine,4-thio-pseudoisocytidine, 4-thio-1-methyl-pseudoisocytidine,4-thio-1-methyl-1-deaza-pseudoisocytidine,1-methyl-1-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine,5-methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine,2-methoxy-cytidine, 2-methoxy-5-methyl-cytidine,4-methoxy-pseudoisocytidine, 4-methoxy-1-methyl-pseudoisocytidine,lysidine (k₂C), α-thio-cytidine, 2′-O-methyl-cytidine (Cm),5,2′-O-dimethyl-cytidine (m⁵Cm), N4-acetyl-2′-O-methyl-cytidine (ac⁴Cm),N4,2′-O-dimethyl-cytidine (m⁴Cm), 5-formyl-2′-O-methyl-cytidine (f⁵Cm),N4,N4,2′-O-trimethyl-cytidine (m⁴²Cm), 1-thio-cytidine,2′-F-ara-cytidine, 2′-F-cytidine, and 2′-OH-ara-cytidine.

In some embodiments, the modified nucleobase is a modified adenine.Exemplary nucleobases and nucleosides having a modified adenine include2-amino-purine, 2, 6-diaminopurine, 2-amino-6-halo-purine (e.g.,2-amino-6-chloro-purine), 6-halo-purine (e.g., 6-chloro-purine),2-amino-6-methyl-purine, 8-azido-adenosine, 7-deaza-adenine,7-deaza-8-aza-adenine, 7-deaza-2-amino-purine,7-deaza-8-aza-2-amino-purine, 7-deaza-2,6-diaminopurine,7-deaza-8-aza-2,6-diaminopurine, 1-methyl-adenosine (m¹A),2-methyl-adenine (m²A), N6-methyl-adenosine (m⁶A),2-methylthio-N6-methyl-adenosine (ms²m6A), N6-isopentenyl-adenosine(i⁶A), 2-methylthio-N6-isopentenyl-adenosine (ms²i⁶A),N6-(cis-hydroxyisopentenyl)adenosine (io⁶A),2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine (ms²io⁶A),N6-glycinylcarbamoyl-adenosine (g⁶A), N6-threonylcarbamoyl-adenosine(t⁶A), N6-methyl-N6-threonylcarbamoyl-adenosine (m⁶t⁶A),2-methylthio-N6-threonylcarbamoyl-adenosine (ms²g⁶A),N6,N6-dimethyl-adenosine (m⁶ ₂A), N6-hydroxynorvalylcarbamoyl-adenosine(hn⁶A), 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine (ms²hn⁶A),N6-acetyl-adenosine (ac⁶A), 7-methyl-adenine, 2-methylthio-adenine,2-methoxy-adenine, α-thio-adenosine, 2′-O-methyl-adenosine (Am),N6,2′-O-dimethyl-adenosine (m⁶Am), N6,N6,2′-O-trimethyl-adenosine (m⁶₂Am), 1,2′-O-dimethyl-adenosine (m Am), 2′-O-ribosyladenosine(phosphate) (Ar(p)), 2-amino-N6-methyl-purine, 1-thio-adenosine,8-azido-adenosine, 2′-F-ara-adenosine, 2′-F-adenosine,2′-OH-ara-adenosine, and N6-(19-amino-pentaoxanonadecyl)-adenosine.

In some embodiments, the modified nucleobase is a modified guanine.Exemplary nucleobases and nucleosides having a modified guanine includeinosine (I), 1-methyl-inosine (m¹I), wyosine (imG), methylwyosine(mimG), 4-demethyl-wyosine (imG-14), isowyosine (imG2), wybutosine (yW),peroxywybutosine (o₂yW), hydroxywybutosine (OhyW), undermodifiedhydroxywybutosine (OhyW*), 7-deaza-guanosine, queuosine (Q),epoxyqueuosine (oQ), galactosyl-queuosine (galQ), mannosyl-queuosine(manQ), 7-cyano-7-deaza-guanosine (preQ₀),7-aminomethyl-7-deaza-guanosine (preQ₁), archaeosine (G⁺),7-deaza-8-aza-guanosine, 6-thio-guanosine, 6-thio-7-deaza-guanosine,6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine (m⁷G),6-thio-7-methyl-guanosine, 7-methyl-inosine, 6-methoxy-guanosine,1-methyl-guanosine (m G), N2-methyl-guanosine (m²G),N2,N2-dimethyl-guanosine (m² ₂G), N2,7-dimethyl-guanosine (m^(2,7)G),N2,N2,7-dimethyl-guanosine (m^(2,2,7)G), 8-oxo-guanosine,7-methyl-8-oxo-guanosine, 1-methyl-6-thio-guanosine,N2-methyl-6-thio-guanosine, N2,N2-dimethyl-6-thio-guanosine,α-thio-guanosine, 2′-O-methyl-guanosine (Gm),N2-methyl-2′-O-methyl-guanosine (m²Gm),N2,N2-dimethyl-2′-O-methyl-guanosine (m² ₂Gm),1-methyl-2′-O-methyl-guanosine (m¹Gm),N2,7-dimethyl-2′-O-methyl-guanosine (m^(2,7)Gm), 2′-O-methyl-inosine(Im), 1,2′-O-dimethyl-inosine (m¹Im), 2′-O-ribosylguanosine (phosphate)(Gr(p)), 1-thio-guanosine, O6-methyl-guanosine, 2′-F-ara-guanosine, and2′-F-guanosine.

In Vitro Transcription of RNA (e.g., mRNA)

VZV vaccines of the present disclosure comprise at least one RNApolynucleotide, such as a mRNA (e.g., modified mRNA). mRNA, for example,is transcribed in vitro from template DNA, referred to as an “in vitrotranscription template.” In some embodiments, the at least one RNApolynucleotide has at least one chemical modification. The at least onechemical modification may include, but is expressly not limited to, anymodification described herein.

In vitro transcription of RNA is known in the art and is described inInternational Publication WO2014/152027, which is incorporated byreference herein in its entirety. For example, in some embodiments, theRNA transcript is generated using a non-amplified, linearized DNAtemplate in an in vitro transcription reaction to generate the RNAtranscript. In some embodiments the RNA transcript is capped viaenzymatic capping. In some embodiments the RNA transcript is purifiedvia chromatographic methods, e.g., use of an oligo dT substrate. Someembodiments exclude the use of DNase. In some embodiments the RNAtranscript is synthesized from a non-amplified, linear DNA templatecoding for the gene of interest via an enzymatic in vitro transcriptionreaction utilizing a T7 phage RNA polymerase and nucleotidetriphosphates of the desired chemistry. Any number of RNA polymerases orvariants may be used in the method of the present invention. Thepolymerase may be selected from, but is not limited to, a phage RNApolymerase, e.g., a T7 RNA polymerase, a T3 RNA polymerase, a SP6 RNApolymerase, and/or mutant polymerases such as, but not limited to,polymerases able to incorporate modified nucleic acids and/or modifiednucleotides, including chemically modified nucleic acids and/ornucleotides.

In some embodiments a non-amplified, linearized plasmid DNA is utilizedas the template DNA for in vitro transcription. In some embodiments, thetemplate DNA is isolated DNA. In some embodiments, the template DNA iscDNA. In some embodiments, the cDNA is formed by reverse transcriptionof a RNA polynucleotide, for example, but not limited to VZV RNA, e.g.VZV mRNA. In some embodiments, Cells, e.g., bacterial cells, e.g., E.coli, e.g., DH-1 cells are transfected with the plasmid DNA template. Insome embodiments, the transfected cells are cultured to replicate theplasmid DNA which is then isolated and purified. In some embodiments,the DNA template includes a RNA polymerase promoter, e.g., a T7 promoterlocated 5′ to and operably linked to the gene of interest.

In some embodiments, an in vitro transcription template encodes a 5′untranslated (UTR) region, contains an open reading frame, and encodes a3′ UTR and a polyA tail. The particular nucleic acid sequencecomposition and length of an in vitro transcription template will dependon the mRNA encoded by the template.

A “5′ untranslated region” (UTR) refers to a region of an mRNA that isdirectly upstream (i.e., 5′) from the start codon (i.e., the first codonof an mRNA transcript translated by a ribosome) that does not encode apolypeptide.

A “3′ untranslated region” (UTR) refers to a region of an mRNA that isdirectly downstream (i.e., 3′) from the stop codon (i.e., the codon ofan mRNA transcript that signals a termination of translation) that doesnot encode a polypeptide.

An “open reading frame” is a continuous stretch of DNA or RNA beginningwith a start codon (e.g., methionine (ATG or AUG)), and ending with astop codon (e.g., TAA, TAG or TGA, or UAA, UAG or UGA) and typicallyencodes a polypeptide (e.g., protein). It will be understood that thesequences disclosed herein may further comprise additional elements,e.g., 5′ and 3′ UTRs, but that those elements, unlike the ORF, need notnecessarily be present in a vaccine of the present disclosure.

A “polyA tail” is a region of mRNA that is downstream, e.g., directlydownstream (i.e., 3′), from the 3′ UTR that contains multiple,consecutive adenosine monophosphates. A polyA tail may contain 10 to 300adenosine monophosphates. For example, a polyA tail may contain 10, 20,30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180,190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 adenosinemonophosphates. In some embodiments, a polyA tail contains 50 to 250adenosine monophosphates. In a relevant biological setting (e.g., incells, in vivo) the poly(A) tail functions to protect mRNA fromenzymatic degradation, e.g., in the cytoplasm, and aids in transcriptiontermination, and/or export of the mRNA from the nucleus and translation.

In some embodiments, a polynucleotide includes 200 to 3,000 nucleotides.For example, a polynucleotide may include 200 to 500, 200 to 1000, 200to 1500, 200 to 3000, 500 to 1000, 500 to 1500, 500 to 2000, 500 to3000, 1000 to 1500, 1000 to 2000, 1000 to 3000, 1500 to 3000, or 2000 to3000 nucleotides).

Methods of Treatment

Provided herein are compositions (e.g., pharmaceutical compositions),methods, kits and reagents for prevention and/or treatment of VZV inhumans and other mammals. VZV RNA vaccines can be used as therapeutic orprophylactic agents. They may be used in medicine to prevent and/ortreat infectious disease. In exemplary aspects, the VZV RNA vaccines ofthe invention are used to provide prophylactic protection from varicellaand herpes zoster. Varicella is an acute infectious disease caused byVZV. The primary varicella zoster virus infection that results inchickenpox (varicella) may result in complications, including viral orsecondary bacterial pneumonia. Even when the clinical symptoms ofchickenpox have resolved, VZV remains dormant in the nervous system ofthe infected person in the trigeminal and dorsal root ganglia and mayreactivate later in life, travelling from the sensory ganglia back tothe skin where it produces a disease (rash) known as shingles or herpeszoster, and can also cause a number of neurologic conditions rangingfrom aseptic meningitis to encephalitis. The VZV vaccines of the presentdisclosure can be used to prevent and/or treat both the primaryinfection (Chicken pox) and also the re-activated viral infection(shingles or herpes zoster) and may be particularly useful forprevention and/or treatment of immunocompromised and elderly patients toprevent or to reduce the severity and/or duration of herpes zoster.

Prophylactic protection from VZV can be achieved followingadministration of a VZV RNA vaccine of the present disclosure. Vaccinescan be administered once, twice, three times, four times or more but itis likely sufficient to administer the vaccine once (optionally followedby a single booster). It is possible, although less desirable, toadminister the vaccine to an infected individual to achieve atherapeutic response. Dosing may need to be adjusted accordingly.

A method of eliciting an immune response in a subject against a VZV isprovided in aspects of the present disclosure. The method involvesadministering to the subject a VZV RNA vaccine comprising at least oneRNA (e.g., mRNA) polynucleotide having an open reading frame encoding atleast one VZV antigenic polypeptide, thereby inducing in the subject animmune response specific to VZV antigenic polypeptide, whereinanti-antigenic polypeptide antibody titer in the subject is increasedfollowing vaccination relative to anti-antigenic polypeptide antibodytiter in a subject vaccinated with a prophylactically effective dose ofa traditional vaccine against the VZV. An “anti-antigenic polypeptideantibody” is a serum antibody the binds specifically to the antigenicpolypeptide.

A prophylactically effective dose is a therapeutically effective dosethat prevents infection with the virus at a clinically acceptable level.In some embodiments, the therapeutically effective dose is a dose listedin a package insert for the vaccine. A traditional vaccine, as usedherein, refers to a vaccine other than the mRNA vaccines of the presentdisclosure. For instance, a traditional vaccine includes, but is notlimited, to live microorganism vaccines, killed microorganism vaccines,subunit vaccines, protein antigen vaccines, DNA vaccines, VLP vaccines,etc. In exemplary embodiments, a traditional vaccine is a vaccine thathas achieved regulatory approval and/or is registered by a national drugregulatory body, for example the Food and Drug Administration (FDA) inthe United States or the European Medicines Agency (EMA).

A method of eliciting an immune response in a subject against a VZV isprovided in aspects of the invention. The method involves administeringto the subject a VZV RNA vaccine comprising at least one RNApolynucleotide having an open reading frame encoding at least one VZVantigenic polypeptide, thereby inducing in the subject an immuneresponse specific to VZV antigenic polypeptide, wherein anti-antigenicpolypeptide antibody titer in the subject is increased followingvaccination relative to anti-antigenic polypeptide antibody titer in asubject vaccinated with a prophylactically effective dose of atraditional vaccine against the VZV. An “anti-antigenic polypeptideantibody” is a serum antibody the binds specifically to the antigenicpolypeptide.

A prophylactically effective dose is a therapeutically effective dosethat prevents infection with the virus at a clinically acceptable level.In some embodiments the therapeutically effective dose is a dose listedin a package insert for the vaccine. A traditional vaccine, as usedherein, refers to a vaccine other than the mRNA vaccines of theinvention. For instance, a traditional vaccine includes but is notlimited to live microorganism vaccines, killed microorganism vaccines,subunit vaccines, protein antigen vaccines, DNA vaccines, etc.

In some embodiments the anti-antigenic polypeptide antibody titer in thesubject is increased 1 log to 10 log following vaccination relative toanti-antigenic polypeptide antibody titer in a subject vaccinated with aprophylactically effective dose of a traditional vaccine against theVZV.

In some embodiments the anti-antigenic polypeptide antibody titer in thesubject is increased 1 log following vaccination relative toanti-antigenic polypeptide antibody titer in a subject vaccinated with aprophylactically effective dose of a traditional vaccine against theVZV.

In some embodiments the anti-antigenic polypeptide antibody titer in thesubject is increased 2 log following vaccination relative toanti-antigenic polypeptide antibody titer in a subject vaccinated with aprophylactically effective dose of a traditional vaccine against theVZV.

In some embodiments the anti-antigenic polypeptide antibody titer in thesubject is increased 3 log following vaccination relative toanti-antigenic polypeptide antibody titer in a subject vaccinated with aprophylactically effective dose of a traditional vaccine against theVZV.

In some embodiments the anti-antigenic polypeptide antibody titer in thesubject is increased 5 log following vaccination relative toanti-antigenic polypeptide antibody titer in a subject vaccinated with aprophylactically effective dose of a traditional vaccine against theVZV.

In some embodiments the anti-antigenic polypeptide antibody titer in thesubject is increased 10 log following vaccination relative toanti-antigenic polypeptide antibody titer in a subject vaccinated with aprophylactically effective dose of a traditional vaccine against theVZV.

A method of eliciting an immune response in a subject against a VZV isprovided in other aspects of the invention. The method involvesadministering to the subject a VZV RNA vaccine comprising at least oneRNA polynucleotide having an open reading frame encoding at least oneVZV antigenic polypeptide, thereby inducing in the subject an immuneresponse specific to VZV antigenic polypeptide, wherein the immuneresponse in the subject is equivalent to an immune response in a subjectvaccinated with a traditional vaccine against the VZV at 2 times to 100times the dosage level relative to the RNA vaccine.

In some embodiments the immune response in the subject is equivalent toan immune response in a subject vaccinated with a traditional vaccine attwice the dosage level relative to the VZV RNA vaccine.

In some embodiments the immune response in the subject is equivalent toan immune response in a subject vaccinated with a traditional vaccine atthree times the dosage level relative to the VZV RNA vaccine.

In some embodiments the immune response in the subject is equivalent toan immune response in a subject vaccinated with a traditional vaccine at4 times the dosage level relative to the VZV RNA vaccine.

In some embodiments the immune response in the subject is equivalent toan immune response in a subject vaccinated with a traditional vaccine at5 times the dosage level relative to the VZV RNA vaccine.

In some embodiments the immune response in the subject is equivalent toan immune response in a subject vaccinated with a traditional vaccine at10 times the dosage level relative to the VZV RNA vaccine.

In some embodiments the immune response in the subject is equivalent toan immune response in a subject vaccinated with a traditional vaccine at50 times the dosage level relative to the VZV RNA vaccine.

In some embodiments the immune response in the subject is equivalent toan immune response in a subject vaccinated with a traditional vaccine at100 times the dosage level relative to the VZV RNA vaccine.

In some embodiments the immune response in the subject is equivalent toan immune response in a subject vaccinated with a traditional vaccine at10 times to 1000 times the dosage level relative to the VZV RNA vaccine.

In some embodiments the immune response in the subject is equivalent toan immune response in a subject vaccinated with a traditional vaccine at100 times to 1000 times the dosage level relative to the VZV RNAvaccine.

In other embodiments the immune response is assessed by determining[protein]antibody titer in the subject.

In other aspects the invention is a method of eliciting an immuneresponse in a subject against a VZV by administering to the subject aVZV RNA vaccine comprising at least one RNA polynucleotide having anopen reading frame encoding at least one VZV antigenic polypeptide,thereby inducing in the subject an immune response specific to VZVantigenic polypeptide, wherein the immune response in the subject isinduced 2 days to 10 weeks earlier relative to an immune responseinduced in a subject vaccinated with a prophylactically effective doseof a traditional vaccine against the VZV. In some embodiments the immuneresponse in the subject is induced in a subject vaccinated with aprophylactically effective dose of a traditional vaccine at 2 times to100 times the dosage level relative to the RNA vaccine.

In some embodiments the immune response in the subject is induced 2 daysearlier relative to an immune response induced in a subject vaccinatedwith a prophylactically effective dose of a traditional vaccine.

In some embodiments the immune response in the subject is induced 3 daysearlier relative to an immune response induced in a subject vaccinated aprophylactically effective dose of a traditional vaccine.

In some embodiments the immune response in the subject is induced 1 weekearlier relative to an immune response induced in a subject vaccinatedwith a prophylactically effective dose of a traditional vaccine.

In some embodiments the immune response in the subject is induced 2weeks earlier relative to an immune response induced in a subjectvaccinated with a prophylactically effective dose of a traditionalvaccine.

In some embodiments the immune response in the subject is induced 3weeks earlier relative to an immune response induced in a subjectvaccinated with a prophylactically effective dose of a traditionalvaccine.

In some embodiments the immune response in the subject is induced 5weeks earlier relative to an immune response induced in a subjectvaccinated with a prophylactically effective dose of a traditionalvaccine.

In some embodiments the immune response in the subject is induced 10weeks earlier relative to an immune response induced in a subjectvaccinated with a prophylactically effective dose of a traditionalvaccine.

A method of v eliciting an immune response in a subject against a VZV byadministering to the subject a VZV RNA vaccine having an open readingframe encoding a first antigenic polypeptide, wherein the RNApolynucleotide does not include a stabilization element, and wherein anadjuvant is not co-formulated or co-administered with the vaccine.

Flagellin Adjuvants

Flagellin is an approximately 500 amino acid monomeric protein thatpolymerizes to form the flagella associated with bacterial motion.Flagellin is expressed by a variety of flagellated bacteria (Salmonellatyphimurium for example) as well as non-flagellated bacteria (such asEscherichia coli). Sensing of flagellin by cells of the innate immunesystem (dendritic cells, macrophages, etc.) is mediated by the Toll-likereceptor 5 (TLR5) as well as by Nod-like receptors (NLRs) Ipaf andNaip5. TLRs and NLRs have been identified as playing a role in theactivation of innate immune response and adaptive immune response. Assuch, flagellin provides an adjuvant effect in a vaccine.

The nucleotide and amino acid sequences encoding known flagellinpolypeptides are publicly available in the NCBI GenBank database. Theflagellin sequences from S. typhimurium, H. pylori, V. cholera, S.marcesens, S. flexneri, T. pallidum, L. pneumophila, B. burgdorferei, C.difficile, R. meliloti, A. tumefaciens, R. lupini, B. clarridgeiae, P.mirabilis, B. subtilus, L. monocytogenes, P. aeruginosa, and E. coli,among others are known.

A flagellin polypeptide, as used herein, refers to a full lengthflagellin protein, immunogenic fragments thereof, and peptides having atleast 50% sequence identify to a flagellin protein or immunogenicfragments thereof. Exemplary flagellin proteins include flagellin fromSalmonella typhi (UniPro Entry number: Q56086), Salmonella typhimurium(AOAOC9DG09), Salmonella enteritidis (AOAOC9BAB7), and Salmonellacholeraesuis (Q6V2X8), and SEQ ID NO: 115-117. In some embodiments, theflagellin polypeptide has at least 60%, 70%, 75%, 80%, 90%, 95%, 97%,98%, or 99% sequence identify to a flagellin protein or immunogenicfragments thereof.

In some embodiments, the flagellin polypeptide is an immunogenicfragment. An immunogenic fragment is a portion of a flagellin proteinthat provokes an immune response. In some embodiments, the immuneresponse is a TLR5 immune response. An example of an immunogenicfragment is a flagellin protein in which all or a portion of a hingeregion has been deleted or replaced with other amino acids. For example,an antigenic polypeptide may be inserted in the hinge region. Hingeregions are the hypervariable regions of a flagellin. Hinge regions of aflagellin are also referred to as “D3 domain or region, “propellerdomain or region,” “hypervariable domain or region” and “variable domainor region.” “At least a portion of a hinge region,” as used herein,refers to any part of the hinge region of the flagellin, or the entiretyof the hinge region. In other embodiments an immunogenic fragment offlagellin is a 20, 25, 30, 35, or 40 amino acid C-terminal fragment offlagellin.

The flagellin monomer is formed by domains D0 through D3. D0 and D1,which form the stem, are composed of tandem long alpha helices and arehighly conserved among different bacteria. The D1 domain includesseveral stretches of amino acids that are useful for TLR5 activation.The entire D1 domain or one or more of the active regions within thedomain are immunogenic fragments of flagellin. Examples of immunogenicregions within the D1 domain include residues 88-114 and residues411-431 (in Salmonella typhimurium FliC flagellin. Within the 13 aminoacids in the 88-100 region, at least 6 substitutions are permittedbetween Salmonella flagellin and other flagellin proteins that stillpreserve TLR5 activation. Thus, immunogenic fragments of flagellininclude flagellin like sequences that activate TLR5 and contain a 13amino acid motif that is 53% or more identical to the Salmonellasequence in 88-100 of FliC (LQRVRELAVQSAN; SEQ ID NO: 118).

In some embodiments, the RNA (e.g., mRNA) vaccine includes an RNA thatencodes a fusion protein of flagellin and one or more antigenicpolypeptides. A “fusion protein” as used herein, refers to a linking oftwo components of the construct. In some embodiments, a carboxy-terminusof the antigenic polypeptide is fused or linked to an amino terminus ofthe flagellin polypeptide. In other embodiments, an amino-terminus ofthe antigenic polypeptide is fused or linked to a carboxy-terminus ofthe flagellin polypeptide. The fusion protein may include, for example,one, two, three, four, five, six or more flagellin polypeptides linkedto one, two, three, four, five, six or more antigenic polypeptides. Whentwo or more flagellin polypeptides and/or two or more antigenicpolypeptides are linked such a construct may be referred to as a“multimer.”

Each of the components of a fusion protein may be directly linked to oneanother or they may be connected through a linker. For instance, thelinker may be an amino acid linker. The amino acid linker encoded for bythe RNA (e.g., mRNA) vaccine to link the components of the fusionprotein may include, for instance, at least one member selected from thegroup consisting of a lysine residue, a glutamic acid residue, a serineresidue and an arginine residue. In some embodiments the linker is 1-30,1-25, 1-25, 5-10, 5, 15, or 5-20 amino acids in length.

In other embodiments the RNA (e.g., mRNA) vaccine includes at least twoseparate RNA polynucleotides, one encoding one or more antigenicpolypeptides and the other encoding the flagellin polypeptide. The atleast two RNA polynucleotides may be co-formulated in a carrier such asa lipid nanoparticle.

Therapeutic and Prophylactic Compositions

Provided herein are compositions (e.g., pharmaceutical compositions),methods, kits and reagents for prevention, treatment or diagnosis of VZVin humans and other mammals, for example. VZV RNA (e.g., mRNA) vaccinescan be used as therapeutic or prophylactic agents. They may be used inmedicine to prevent and/or treat infectious disease. In someembodiments, the VZV vaccines of the invention can be envisioned for usein the priming of immune effector cells, for example, to activateperipheral blood mononuclear cells (PBMCs) ex vivo, which are theninfused (re-infused) into a subject.

In exemplary embodiments, a VZV vaccine containing RNA polynucleotidesas described herein can be administered to a subject (e.g., a mammaliansubject, such as a human subject), and the RNA polynucleotides aretranslated in vivo to produce an antigenic polypeptide.

The VZV RNA vaccines may be induced for translation of a polypeptide(e.g., antigen or immunogen) in a cell, tissue or organism. In exemplaryembodiments, such translation occurs in vivo, although there can beenvisioned embodiments where such translation occurs ex vivo, in cultureor in vitro. In exemplary embodiments, the cell, tissue or organism iscontacted with an effective amount of a composition containing a VZV RNAvaccine that contains a polynucleotide that has at least one atranslatable region encoding an antigenic polypeptide.

An “effective amount” of the VZV RNA vaccine is provided based, at leastin part, on the target tissue, target cell type, means ofadministration, physical characteristics of the polynucleotide (e.g.,size, and extent of modified nucleosides) and other components of theVZV RNA vaccine, and other determinants. In general, an effective amountof the VZV RNA vaccine composition provides an induced or boosted immuneresponse as a function of antigen production in the cell. In general, aneffective amount of the VZV RNA vaccine containing RNA polynucleotideshaving at least one chemical modifications are preferably more efficientthan a composition containing a corresponding unmodified polynucleotideencoding the same antigen or a peptide antigen. Increased antigenproduction may be demonstrated by increased cell transfection (thepercentage of cells transfected with the RNA vaccine), increased proteintranslation from the polynucleotide, decreased nucleic acid degradation(as demonstrated, for example, by increased duration of proteintranslation from a modified polynucleotide), or altered antigen specificimmune response of the host cell.

The term “pharmaceutical composition” refers to the combination of anactive agent with a carrier, inert or active, making the compositionespecially suitable for diagnostic or therapeutic use in vivo or exvivo. A “pharmaceutically acceptable carrier,” after administered to orupon a subject, does not cause undesirable physiological effects. Thecarrier in the pharmaceutical composition must be “acceptable” also inthe sense that it is compatible with the active ingredient and can becapable of stabilizing it. One or more solubilizing agents can beutilized as pharmaceutical carriers for delivery of an active agent.Examples of a pharmaceutically acceptable carrier include, but are notlimited to, biocompatible vehicles, adjuvants, additives, and diluentsto achieve a composition usable as a dosage form. Examples of othercarriers include colloidal silicon oxide, magnesium stearate, cellulose,and sodium lauryl sulfate. Additional suitable pharmaceutical carriersand diluents, as well as pharmaceutical necessities for their use, aredescribed in Remington's Pharmaceutical Sciences.

In some embodiments, RNA vaccines (including polynucleotides and theirencoded polypeptides) in accordance with the present disclosure may beused for treatment or prevention of VZV.

VZV RNA vaccines may be administered prophylactically or therapeuticallyas part of an active immunization scheme to healthy individuals or earlyin infection during the incubation phase or during active infectionafter onset of symptoms. In some embodiments, the amount of RNA vaccinesof the present disclosure provided to a cell, a tissue or a subject maybe an amount effective for immune prophylaxis.

VZV RNA (e.g., mRNA) vaccines may be administrated with otherprophylactic or therapeutic compounds. As a non-limiting example, aprophylactic or therapeutic compound may be an adjuvant or a booster. Asused herein, when referring to a prophylactic composition, such as avaccine, the term “booster” refers to an extra administration of theprophylactic (vaccine) composition. A booster (or booster vaccine) maybe given after an earlier administration of the prophylacticcomposition. The time of administration between the initialadministration of the prophylactic composition and the booster may be,but is not limited to, 1 minute, 2 minutes, 3 minutes, 4 minutes, 5minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 15minutes, 20 minutes 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8hours, 9 hours, 10 hours, 11 hours, 12 hours, 13 hours, 14 hours, 15hours, 16 hours, 17 hours, 18 hours, 19 hours, 20 hours, 21 hours, 22hours, 23 hours, 1 day, 36 hours, 2 days, 3 days, 4 days, 5 days, 6days, 1 week, 10 days, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, 4months, 5 months, 6 months, 7 months, 8 months, 9 months, 10 months, 11months, 1 year, 18 months, 2 years, 3 years, 4 years, 5 years, 6 years,7 years, 8 years, 9 years, 10 years, 11 years, 12 years, 13 years, 14years, 15 years, 16 years, 17 years, 18 years, 19 years, 20 years, 25years, 30 years, 35 years, 40 years, 45 years, 50 years, 55 years, 60years, 65 years, 70 years, 75 years, 80 years, 85 years, 90 years, 95years or more than 99 years. In exemplary embodiments, the time ofadministration between the initial administration of the prophylacticcomposition and the booster may be, but is not limited to, 1 week, 2weeks, 3 weeks, 1 month, 2 months, 3 months, 6 months or 1 year.

In some embodiments, VZV RNA vaccines may be administeredintramuscularly, intranasally or intradermally, similarly to theadministration of inactivated vaccines known in the art.

The VZV RNA vaccines may be utilized in various settings depending onthe prevalence of the infection or the degree or level of unmet medicalneed. As a non-limiting example, the RNA vaccines may be utilized totreat and/or prevent a variety of infectious disease. RNA vaccines havesuperior properties in that they produce much larger antibody titers andproduce responses early than commercially available anti-virals.

Provided herein are pharmaceutical compositions including VZV RNAvaccines and RNA vaccine compositions and/or complexes optionally incombination with one or more pharmaceutically acceptable excipients.

VZV RNA (e.g., mRNA) vaccines may be formulated or administered alone orin conjunction with one or more other components. For instance, VZV RNAvaccines (vaccine compositions) may comprise other components including,but not limited to, adjuvants.

In some embodiments, VZV RNA vaccines do not include an adjuvant (theyare adjuvant free).

VZV RNA (e.g., mRNA) vaccines may be formulated or administered incombination with one or more pharmaceutically-acceptable excipients. Insome embodiments, vaccine compositions comprise at least one additionalactive substances, such as, for example, a therapeutically-activesubstance, a prophylactically-active substance, or a combination ofboth. Vaccine compositions may be sterile, pyrogen-free or both sterileand pyrogen-free. General considerations in the formulation and/ormanufacture of pharmaceutical agents, such as vaccine compositions, maybe found, for example, in Remington: The Science and Practice ofPharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporatedherein by reference in its entirety).

In some embodiments, VZV RNA vaccines are administered to humans, humanpatients or subjects. For the purposes of the present disclosure, thephrase “active ingredient” generally refers to the RNA vaccines or thepolynucleotides contained therein, for example, RNA polynucleotides(e.g., mRNA polynucleotides) encoding antigenic polypeptides.

Formulations of the vaccine compositions described herein may beprepared by any method known or hereafter developed in the art ofpharmacology. In general, such preparatory methods include the step ofbringing the active ingredient (e.g., mRNA polynucleotide) intoassociation with an excipient and/or one or more other accessoryingredients, and then, if necessary and/or desirable, dividing, shapingand/or packaging the product into a desired single- or multi-dose unit.

Relative amounts of the active ingredient, the pharmaceuticallyacceptable excipient, and/or any additional ingredients in apharmaceutical composition in accordance with the disclosure will vary,depending upon the identity, size, and/or condition of the subjecttreated and further depending upon the route by which the composition isto be administered. By way of example, the composition may comprisebetween 0.1% and 100%, e.g., between 0.5 and 50%, between 1-30%, between5-80%, at least 80% (w/w) active ingredient.

VZV RNA vaccines can be formulated using one or more excipients to: (1)increase stability; (2) increase cell transfection; (3) permit thesustained or delayed release (e.g., from a depot formulation); (4) alterthe biodistribution (e.g., target to specific tissues or cell types);(5) increase the translation of encoded protein in vivo; and/or (6)alter the release profile of encoded protein (antigen) in vivo. Inaddition to traditional excipients such as any and all solvents,dispersion media, diluents, or other liquid vehicles, dispersion orsuspension aids, surface active agents, isotonic agents, thickening oremulsifying agents, preservatives, excipients can include, withoutlimitation, lipidoids, liposomes, lipid nanoparticles, polymers,lipoplexes, core-shell nanoparticles, peptides, proteins, cellstransfected with VZV RNA vaccines (e.g., for transplantation into asubject), hyaluronidase, nanoparticle mimics and combinations thereof.

Stabilizing Elements

Naturally-occurring eukaryotic mRNA molecules have been found to containstabilizing elements, including, but not limited to untranslated regions(UTR) at their 5′-end (5′UTR) and/or at their 3′-end (3′UTR), inaddition to other structural features, such as a 5′-cap structure or a3′-poly(A) tail. Both the 5′UTR and the 3′UTR are typically transcribedfrom the genomic DNA and are elements of the premature mRNA.Characteristic structural features of mature mRNA, such as the 5′-capand the 3′-poly(A) tail are usually added to the transcribed (premature)mRNA during mRNA processing. The 3′-poly(A) tail is typically a stretchof adenine nucleotides added to the 3′-end of the transcribed mRNA. Itcan comprise up to about 400 adenine nucleotides. In some embodimentsthe length of the 3′-poly(A) tail may be an essential element withrespect to the stability of the individual mRNA.

In some embodiments the RNA vaccine may include one or more stabilizingelements. Stabilizing elements may include for instance a histonestem-loop. A stem-loop binding protein (SLBP), a 32 kDa protein has beenidentified. It is associated with the histone stem-loop at the 3′-end ofthe histone messages in both the nucleus and the cytoplasm. Itsexpression level is regulated by the cell cycle; it peaks during theS-phase, when histone mRNA levels are also elevated. The protein hasbeen shown to be essential for efficient 3′-end processing of histonepre-mRNA by the U7 snRNP. SLBP continues to be associated with thestem-loop after processing, and then stimulates the translation ofmature histone mRNAs into histone proteins in the cytoplasm. The RNAbinding domain of SLBP is conserved through metazoa and protozoa; itsbinding to the histone stem-loop depends on the structure of the loop.The minimum binding site includes at least three nucleotides 5′ and twonucleotides 3′ relative to the stem-loop.

In some embodiments, the RNA vaccines include a coding region, at leastone histone stem-loop, and optionally, a poly(A) sequence orpolyadenylation signal. The poly(A) sequence or polyadenylation signalgenerally should enhance the expression level of the encoded protein.The encoded protein, in some embodiments, is not a histone protein, areporter protein (e.g. Luciferase, GFP, EGFP, β-Galactosidase, EGFP), ora marker or selection protein (e.g. alpha-Globin, Galactokinase andXanthine:guanine phosphoribosyl transferase (GPT)).

In some embodiments, the combination of a poly(A) sequence orpolyadenylation signal and at least one histone stem-loop, even thoughboth represent alternative mechanisms in nature, acts synergistically toincrease the protein expression beyond the level observed with either ofthe individual elements. It has been found that the synergistic effectof the combination of poly(A) and at least one histone stem-loop doesnot depend on the order of the elements or the length of the poly(A)sequence.

In some embodiments, the RNA vaccine does not comprise a histonedownstream element (HDE). “Histone downstream element” (HDE) includes apurine-rich polynucleotide stretch of approximately 15 to 20 nucleotides3′ of naturally occurring stem-loops, representing the binding site forthe U7 snRNA, which is involved in processing of histone pre-mRNA intomature histone mRNA. In some embodiments, the nucleic acid does notinclude an intron.

In some embodiments, the RNA vaccine may or may not contain a enhancerand/or promoter sequence, which may be modified or unmodified or whichmay be activated or inactivated. In some embodiments, the histonestem-loop is generally derived from histone genes, and includes anintramolecular base pairing of two neighbored partially or entirelyreverse complementary sequences separated by a spacer, consisting of ashort sequence, which forms the loop of the structure. The unpaired loopregion is typically unable to base pair with either of the stem loopelements. It occurs more often in RNA, as is a key component of many RNAsecondary structures, but may be present in single-stranded DNA as well.Stability of the stem-loop structure generally depends on the length,number of mismatches or bulges, and base composition of the pairedregion. In some embodiments, wobble base pairing (non-Watson-Crick basepairing) may result. In some embodiments, the at least one histonestem-loop sequence comprises a length of 15 to 45 nucleotides.

In other embodiments the RNA vaccine may have one or more AU-richsequences removed. These sequences, sometimes referred to as AURES aredestabilizing sequences found in the 3′UTR. The AURES may be removedfrom the RNA vaccines. Alternatively the AURES may remain in the RNAvaccine.

Nanoparticle Formulations

In some embodiments, VZV RNA (e.g., mRNA) vaccines are formulated in ananoparticle. In some embodiments, VZV RNA vaccines are formulated in alipid nanoparticle. In some embodiments, VZV RNA vaccines are formulatedin a lipid-polycation complex, referred to as a cationic lipidnanoparticle. The formation of the lipid nanoparticle may beaccomplished by methods known in the art and/or as described in U.S.Publication No. 2012/0178702, herein incorporated by reference in itsentirety. As a non-limiting example, the polycation may include acationic peptide or a polypeptide such as, but not limited to,polylysine, polyornithine and/or polyarginine and the cationic peptidesdescribed in International Publication No. WO2012/013326 or U.S.Publication No. US2013/0142818; each of which is herein incorporated byreference in its entirety. In some embodiments, VZV RNA vaccines areformulated in a lipid nanoparticle that includes a non-cationic lipidsuch as, but not limited to, cholesterol or dioleoylphosphatidylethanolamine (DOPE).

A lipid nanoparticle formulation may be influenced by, but not limitedto, the selection of the cationic lipid component, the degree ofcationic lipid saturation, the nature of the PEGylation, ratio of allcomponents and biophysical parameters such as size. In one example bySemple et al. (Nature Biotech. 2010 28:172-176; herein incorporated byreference in its entirety), the lipid nanoparticle formulation iscomposed of 57.1% cationic lipid, 7.1% dipalmitoylphosphatidylcholine,34.3% cholesterol, and 1.4% PEG-c-DMA. As another example, changing thecomposition of the cationic lipid was shown to more effectively deliversiRNA to various antigen presenting cells (Basha et al. Mol Ther. 201119:2186-2200; herein incorporated by reference in its entirety).

In some embodiments, lipid nanoparticle formulations may comprise 35 to45% cationic lipid, 40% to 50% cationic lipid, 50% to 60% cationic lipidand/or 55% to 65% cationic lipid. In some embodiments, the ratio oflipid to RNA (e.g., mRNA) in lipid nanoparticles may be 5:1 to 20:1,10:1 to 25:1, 15:1 to 30:1 and/or at least 30:1.

In some embodiments, the ratio of PEG in the lipid nanoparticleformulations may be increased or decreased and/or the carbon chainlength of the PEG lipid may be modified from C14 to C18 to alter thepharmacokinetics and/or biodistribution of the lipid nanoparticleformulations. As a non-limiting example, lipid nanoparticle formulationsmay contain 0.5% to 3.0%, 1.0% to 3.5%, 1.5% to 4.0%, 2.0% to 4.5%, 2.5%to 5.0% and/or 3.0% to 6.0% of the lipid molar ratio of PEG-c-DOMG(R-3-[(co-methoxy-poly(ethyleneglycol)2000)carbamoyl)]-1,2-dimyristyloxypropyl-3-amine)(also referred to herein as PEG-DOMG) as compared to the cationic lipid,DSPC and cholesterol. In some embodiments, the PEG-c-DOMG may bereplaced with a PEG lipid such as, but not limited to, PEG-DSG(1,2-Distearoyl-sn-glycerol, methoxypolyethylene glycol), PEG-DMG(1,2-Dimyristoyl-sn-glycerol) and/or PEG-DPG(1,2-Dipalmitoyl-sn-glycerol, methoxypolyethylene glycol). The cationiclipid may be selected from any lipid known in the art such as, but notlimited to, DLin-MC3-DMA, DLin-DMA, C12-200 and DLin-KC2-DMA.

In some embodiments, a VZV RNA (e.g., mRNA) vaccine formulation is ananoparticle that comprises at least one lipid. The lipid may beselected from, but is not limited to, DLin-DMA, DLin-K-DMA, 98N12-5,C12-200, DLin-MC3-DMA, DLin-KC2-DMA, DODMA, PLGA, PEG, PEG-DMG,(12Z,15Z)—N,N-dimethyl-2-nonylhenicosa-12,15-dien-1-amine,N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]heptadecan-8-amine, PEGylatedlipids and amino alcohol lipids.

In some embodiments, the cationic lipid is

In some embodiments, the cationic lipid is

In some embodiments, the lipid may be a cationic lipid such as, but notlimited to, DLin-DMA, DLin-D-DMA, DLin-MC3-DMA, DLin-KC2-DMA, DODMA andamino alcohol lipids. The amino alcohol cationic lipid may be the lipidsdescribed in and/or made by the methods described in U.S. PublicationNo. US2013/0150625, herein incorporated by reference in its entirety. Asa non-limiting example, the cationic lipid may be2-amino-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-{[(9Z,2Z)-octadeca-9,12-dien-1-yloxy]methyl}propan-1-ol(Compound 1 in US2013/0150625);2-amino-3-[(9Z)-octadec-9-en-1-yloxy]-2-{[(9Z)-octadec-9-en-1-yloxy]methyl}propan-1-ol(Compound 2 in US2013/0150625);2-amino-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-[(octyloxy)methyl]propan-1-ol(Compound 3 in US2013/0150625); and2-(dimethylamino)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-{[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]methyl}propan-1-ol(Compound 4 in US2013/0150625); or any pharmaceutically acceptable saltor stereoisomer thereof.

Lipid nanoparticle formulations typically comprise a lipid, inparticular, an ionizable cationic lipid, for example,2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethyl aminobutyrate (DLin-MC3-DMA), ordi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate, and furthercomprise a neutral lipid, a sterol and a molecule capable of reducingparticle aggregation, for example a PEG or PEG-modified lipid.

In some embodiments, a lipid nanoparticle formulation consistsessentially of (i) at least one lipid selected from the group consistingof 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate; (ii) a neutrallipid selected from DSPC, DPPC, POPC, DOPE and SM; (iii) a sterol, e.g.,cholesterol; and (iv) a PEG-lipid, e.g., PEG-DMG or PEG-cDMA, in a molarratio of 20-60% cationic lipid: 5-25% neutral lipid (non-cationiclipid): 25-55% sterol; 0.5-15% PEG-lipid.

In some embodiments, a lipid nanoparticle formulation includes 25% to75% on a molar basis of a cationic lipid selected from the groupconsisting of 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane(DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate(DLin-MC3-DMA), and di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate, e.g., 35 to 65%, 45to 65%, 60%, 57.5%, 50% or 40% on a molar basis.

In some embodiments, a lipid nanoparticle formulation includes 0.5% to15% on a molar basis of the neutral lipid, e.g., 3 to 12%, 5 to 10% or15%, 10%, or 7.5% on a molar basis. Examples of neutral lipids include,without limitation, DSPC, POPC, DPPC, DOPE and SM. In some embodiments,the formulation includes 5% to 50% on a molar basis of the sterol (e.g.,15 to 45%, 20 to 40%, 40%, 38.5%, 35%, or 31% on a molar basis. Anon-limiting example of a sterol is cholesterol. In some embodiments, alipid nanoparticle formulation includes 0.5% to 20% on a molar basis ofthe PEG or PEG-modified lipid (e.g., 0.5 to 10%, 0.5 to 5%, 1.5%, 0.5%,1.5%, 3.5%, or 5% on a molar basis. In some embodiments, a PEG or PEGmodified lipid comprises a PEG molecule of an average molecular weightof 2,000 Da. In some embodiments, a PEG or PEG modified lipid comprisesa PEG molecule of an average molecular weight of less than 2,000, forexample around 1,500 Da, around 1,000 Da, or around 500 Da. Non-limitingexamples of PEG-modified lipids include PEG-distearoyl glycerol(PEG-DMG) (also referred herein as PEG-C14 or C14-PEG), PEG-cDMA(further discussed in Reyes et al. J. Controlled Release, 107, 276-287(2005) the content of which is herein incorporated by reference in itsentirety).

In some embodiments, lipid nanoparticle formulations include 25-75% of acationic lipid selected from the group consisting of2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate, 0.5-15% of theneutral lipid, 5-50% of the sterol, and 0.5-20% of the PEG orPEG-modified lipid on a molar basis.

In some embodiments, lipid nanoparticle formulations include 35-65% of acationic lipid selected from the group consisting of2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate, 3-12% of theneutral lipid, 15-45% of the sterol, and 0.5-10% of the PEG orPEG-modified lipid on a molar basis.

In some embodiments, lipid nanoparticle formulations include 45-65% of acationic lipid selected from the group consisting of2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate, 5-10% of theneutral lipid, 25-40% of the sterol, and 0.5-10% of the PEG orPEG-modified lipid on a molar basis.

In some embodiments, lipid nanoparticle formulations include 60% of acationic lipid selected from the group consisting of2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate, 7.5% of the neutrallipid, 31% of the sterol, and 1.5% of the PEG or PEG-modified lipid on amolar basis.

In some embodiments, lipid nanoparticle formulations include 50% of acationic lipid selected from the group consisting of2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate, 10% of the neutrallipid, 38.5% of the sterol, and 1.5% of the PEG or PEG-modified lipid ona molar basis.

In some embodiments, lipid nanoparticle formulations include 50% of acationic lipid selected from the group consisting of2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate, 10% of the neutrallipid, 35% of the sterol, 4.5% or 5% of the PEG or PEG-modified lipid,and 0.5% of the targeting lipid on a molar basis.

In some embodiments, lipid nanoparticle formulations include 40% of acationic lipid selected from the group consisting of2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate, 15% of the neutrallipid, 40% of the sterol, and 5% of the PEG or PEG-modified lipid on amolar basis.

In some embodiments, lipid nanoparticle formulations include 57.2% of acationic lipid selected from the group consisting of2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA),dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), anddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate, 7.1% of the neutrallipid, 34.3% of the sterol, and 1.4% of the PEG or PEG-modified lipid ona molar basis.

In some embodiments, lipid nanoparticle formulations include 57.5% of acationic lipid selected from the PEG lipid is PEG-cDMA (PEG-cDMA isfurther discussed in Reyes et al. (J. Controlled Release, 107, 276-287(2005), the content of which is herein incorporated by reference in itsentirety), 7.5% of the neutral lipid, 31.5% of the sterol, and 3.5% ofthe PEG or PEG-modified lipid on a molar basis.

In some embodiments, lipid nanoparticle formulations consistsessentially of a lipid mixture in molar ratios of 20-70% cationic lipid:5-45% neutral lipid: 20-55% cholesterol: 0.5-15% PEG-modified lipid. Insome embodiments, lipid nanoparticle formulations consists essentiallyof a lipid mixture in a molar ratio of 20-60% cationic lipid: 5-25%neutral lipid (non-cationic lipid): 25-55% cholesterol: 0.5-15%PEG-modified lipid.

In some embodiments, the molar lipid ratio is 50/10/38.5/1.5 (mol %cationic lipid/neutral lipid, e.g., DSPC/Chol/PEG-modified lipid, e.g.,PEG-DMG, PEG-DSG or PEG-DPG), 57.2/7.1134.3/1.4 (mol % cationiclipid/neutral lipid, e.g., DPPC/Chol/PEG-modified lipid, e.g.,PEG-cDMA), 40/15/40/5 (mol % cationic lipid/neutral lipid, e.g.,DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG), 50/10/35/4.5/0.5 (mol %cationic lipid/neutral lipid, e.g., DSPC/Chol/PEG-modified lipid, e.g.,PEG-DSG), 50/10/35/5 (cationic lipid/neutral lipid, e.g.,DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG), 40/10/40/10 (mol %cationic lipid/neutral lipid, e.g., DSPC/Chol/PEG-modified lipid, e.g.,PEG-DMG or PEG-cDMA), 35/15/40/10 (mol % cationic lipid/neutral lipid,e.g., DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG or PEG-cDMA) or52/13/30/5 (mol % cationic lipid/neutral lipid, e.g.,DSPC/Chol/PEG-modified lipid, e.g., PEG-DMG or PEG-cDMA).

Non-limiting examples of lipid nanoparticle compositions and methods ofmaking them are described, for example, in Semple et al. (2010) Nat.Biotechnol. 28:172-176; Jayarama et al. (2012), Angew. Chem. Int. Ed.,51: 8529-8533; and Maier et al. (2013) Molecular Therapy 21, 1570-1578(the contents of each of which are incorporated herein by reference intheir entirety).

In some embodiments, lipid nanoparticle formulations may comprise acationic lipid, a PEG lipid and a structural lipid and optionallycomprise a non-cationic lipid. As a non-limiting example, a lipidnanoparticle may comprise 40-60% of cationic lipid, 5-15% of anon-cationic lipid, 1-2% of a PEG lipid and 30-50% of a structurallipid. As another non-limiting example, the lipid nanoparticle maycomprise 50% cationic lipid, 10% non-cationic lipid, 1.5% PEG lipid and38.5% structural lipid. As yet another non-limiting example, a lipidnanoparticle may comprise 55% cationic lipid, 10% non-cationic lipid,2.5% PEG lipid and 32.5% structural lipid. In some embodiments, thecationic lipid may be any cationic lipid described herein such as, butnot limited to, DLin-KC2-DMA, DLin-MC3-DMA and di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate.

In some embodiments, the lipid nanoparticle formulations describedherein may be 4 component lipid nanoparticles. The lipid nanoparticlemay comprise a cationic lipid, a non-cationic lipid, a PEG lipid and astructural lipid. As a non-limiting example, the lipid nanoparticle maycomprise 40-60% of cationic lipid, 5-15% of a non-cationic lipid, 1-2%of a PEG lipid and 30-50% of a structural lipid. As another non-limitingexample, the lipid nanoparticle may comprise 50% cationic lipid, 10%non-cationic lipid, 1.5% PEG lipid and 38.5% structural lipid. As yetanother non-limiting example, the lipid nanoparticle may comprise 55%cationic lipid, 10% non-cationic lipid, 2.5% PEG lipid and 32.5%structural lipid. In some embodiments, the cationic lipid may be anycationic lipid described herein such as, but not limited to,DLin-KC2-DMA, DLin-MC3-DMA and di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate.

In some embodiments, the lipid nanoparticle formulations describedherein may comprise a cationic lipid, a non-cationic lipid, a PEG lipidand a structural lipid. As a non-limiting example, the lipidnanoparticle comprise 50% of the cationic lipid DLin-KC2-DMA, 10% of thenon-cationic lipid DSPC, 1.5% of the PEG lipid PEG-DOMG and 38.5% of thestructural lipid cholesterol. As a non-limiting example, the lipidnanoparticle comprise 50% of the cationic lipid DLin-MC3-DMA, 10% of thenon-cationic lipid DSPC, 1.5% of the PEG lipid PEG-DOMG and 38.5% of thestructural lipid cholesterol. As a non-limiting example, the lipidnanoparticle comprise 50% of the cationic lipid DLin-MC3-DMA, 10% of thenon-cationic lipid DSPC, 1.5% of the PEG lipid PEG-DMG and 38.5% of thestructural lipid cholesterol. As yet another non-limiting example, thelipid nanoparticle comprise 55% of the cationic lipiddi((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate, 10% of thenon-cationic lipid DSPC, 2.5% of the PEG lipid PEG-DMG and 32.5% of thestructural lipid cholesterol.

Relative amounts of the active ingredient, the pharmaceuticallyacceptable excipient, and/or any additional ingredients in a vaccinecomposition may vary, depending upon the identity, size, and/orcondition of the subject being treated and further depending upon theroute by which the composition is to be administered. For example, thecomposition may comprise between 0.1% and 99% (w/w) of the activeingredient. By way of example, the composition may comprise between 0.1%and 100%, e.g., between 0.5 and 50%, between 1-30%, between 5-80%, atleast 80% (w/w) active ingredient.

In some embodiments, the RNA vaccine composition may comprise thepolynucleotide described herein, formulated in a lipid nanoparticlecomprising MC3, Cholesterol, DSPC and PEG2000-DMG, the buffer trisodiumcitrate, sucrose and water for injection. As a non-limiting example, thecomposition comprises: 2.0 mg/mL of drug substance (e.g.,polynucleotides encoding VZV), 21.8 mg/mL of MC3, 10.1 mg/mL ofcholesterol, 5.4 mg/mL of DSPC, 2.7 mg/mL of PEG2000-DMG, 5.16 mg/mL oftrisodium citrate, 71 mg/mL of sucrose and 1.0 mL of water forinjection.

In some embodiments, a nanoparticle (e.g., a lipid nanoparticle) has amean diameter of 10-500 nm, 20-400 nm, 30-300 nm, 40-200 nm. In someembodiments, a nanoparticle (e.g., a lipid nanoparticle) has a meandiameter of 50-150 nm, 50-200 nm, 80-100 nm or 80-200 nm.

Liposomes, Lipoplexes, and Lipid Nanoparticles

In some embodiments, the RNA vaccine pharmaceutical compositions may beformulated in liposomes such as, but not limited to, DiLa2 liposomes(Marina Biotech, Bothell, Wash.), SMARTICLES® (Marina Biotech, Bothell,Wash.), neutral DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) basedliposomes (e.g., siRNA delivery for ovarian cancer (Landen et al. CancerBiology & Therapy 2006 5(12)1708-1713); herein incorporated by referencein its entirety) and hyaluronan-coated liposomes (Quiet Therapeutics,Israel).

In some embodiments, the RNA vaccines may be formulated in a lyophilizedgel-phase liposomal composition as described in U.S. Publication No.US2012/0060293, herein incorporated by reference in its entirety.

The nanoparticle formulations may comprise a phosphate conjugate. Thephosphate conjugate may increase in vivo circulation times and/orincrease the targeted delivery of the nanoparticle. Phosphate conjugatesfor use with the present invention may be made by the methods describedin International Publication No. WO2013/033438 or U.S. Publication No.US2013/0196948, the content of each of which is herein incorporated byreference in its entirety. As a non-limiting example, the phosphateconjugates may include a compound of any one of the formulas describedin International Publication No. WO2013/033438, herein incorporated byreference in its entirety.

The nanoparticle formulation may comprise a polymer conjugate. Thepolymer conjugate may be a water soluble conjugate. The polymerconjugate may have a structure as described in U.S. Publication No.2013/0059360, the content of which is herein incorporated by referencein its entirety. In some aspects, polymer conjugates with thepolynucleotides of the present invention may be made using the methodsand/or segmented polymeric reagents described in U.S. Publication No.2013/0072709, herein incorporated by reference in its entirety. In otheraspects, the polymer conjugate may have pendant side groups comprisingring moieties such as, but not limited to, the polymer conjugatesdescribed in U.S. Publication No. US2013/0196948, the contents of whichis herein incorporated by reference in its entirety.

The nanoparticle formulations may comprise a conjugate to enhance thedelivery of nanoparticles of the present invention in a subject.Further, the conjugate may inhibit phagocytic clearance of thenanoparticles in a subject. In some aspects, the conjugate may be a“self” peptide designed from the human membrane protein CD47 (e.g., the“self” particles described by Rodriguez et al (Science 2013, 339,971-975), herein incorporated by reference in its entirety). As shown byRodriguez et al. the self peptides delayed macrophage-mediated clearanceof nanoparticles which enhanced delivery of the nanoparticles. In otheraspects, the conjugate may be the membrane protein CD47 (e.g., seeRodriguez et al. Science 2013, 339, 971-975, herein incorporated byreference in its entirety). Rodriguez et al. showed that, similarly to“self” peptides, CD47 can increase the circulating particle ratio in asubject as compared to scrambled peptides and PEG coated nanoparticles.

In some embodiments, the RNA vaccines of the present invention areformulated in nanoparticles which comprise a conjugate to enhance thedelivery of the nanoparticles of the present invention in a subject. Theconjugate may be the CD47 membrane or the conjugate may be derived fromthe CD47 membrane protein, such as the “self” peptide describedpreviously. In other embodiments, the nanoparticle may comprise PEG anda conjugate of CD47 or a derivative thereof. In yet other embodiments,the nanoparticle may comprise both the “self” peptide described aboveand the membrane protein CD47.

In some embodiments, a “self” peptide and/or CD47 protein may beconjugated to a virus-like particle or pseudovirion, as described hereinfor delivery of the RNA vaccines of the present invention.

In other embodiments, RNA vaccine pharmaceutical compositions comprisingthe polynucleotides of the present invention and a conjugate, which mayhave a degradable linkage. Non-limiting examples of conjugates includean aromatic moiety comprising an ionizable hydrogen atom, a spacermoiety, and a water-soluble polymer. As a non-limiting example,pharmaceutical compositions comprising a conjugate with a degradablelinkage and methods for delivering such pharmaceutical compositions aredescribed in U.S. Publication No. US2013/0184443, the content of whichis herein incorporated by reference in its entirety.

The nanoparticle formulations may be a carbohydrate nanoparticlecomprising a carbohydrate carrier and a RNA vaccine. As a non-limitingexample, the carbohydrate carrier may include, but is not limited to, ananhydride-modified phytoglycogen or glycogen-type material,phytoglycogen octenyl succinate, phytoglycogen beta-dextrin,anhydride-modified phytoglycogen beta-dextrin. (See e.g., InternationalPublication No. WO2012/109121, the content of which is hereinincorporated by reference in its entirety).

Nanoparticle formulations of the present invention may be coated with asurfactant or polymer in order to improve the delivery of the particle.In some embodiments, the nanoparticle may be coated with a hydrophiliccoating such as, but not limited to, PEG coatings and/or coatings thathave a neutral surface charge. The hydrophilic coatings may help todeliver nanoparticles with larger payloads such as, but not limited to,RNA vaccines within the central nervous system. As a non-limitingexample nanoparticles comprising a hydrophilic coating and methods ofmaking such nanoparticles are described in U.S. Publication No.US2013/0183244, the content of which is herein incorporated by referencein its entirety.

In some embodiments, the lipid nanoparticles of the present inventionmay be hydrophilic polymer particles. Non-limiting examples ofhydrophilic polymer particles and methods of making hydrophilic polymerparticles are described in U.S. Publication No. US2013/0210991, thecontent of which is herein incorporated by reference in its entirety.

In other embodiments, the lipid nanoparticles of the present inventionmay be hydrophobic polymer particles.

Lipid nanoparticle formulations may be improved by replacing thecationic lipid with a biodegradable cationic lipid which is known as arapidly eliminated lipid nanoparticle (reLNP). Ionizable cationiclipids, such as, but not limited to, DLinDMA, DLin-KC2-DMA, andDLin-MC3-DMA, have been shown to accumulate in plasma and tissues overtime and may be a potential source of toxicity. The rapid metabolism ofthe rapidly eliminated lipids can improve the tolerability andtherapeutic index of the lipid nanoparticles by an order of magnitudefrom a 1 mg/kg dose to a 10 mg/kg dose in rat. Inclusion of anenzymatically degraded ester linkage can improve the degradation andmetabolism profile of the cationic component, while still maintainingthe activity of the reLNP formulation. The ester linkage can beinternally located within the lipid chain or it may be terminallylocated at the terminal end of the lipid chain. The internal esterlinkage may replace any carbon in the lipid chain.

In some embodiments, the internal ester linkage may be located on eitherside of the saturated carbo.

In some embodiments, an immune response may be elicited by delivering alipid nanoparticle which may include a nanospecies, a polymer and animmunogen. (U.S. Publication No. 2012/0189700 and InternationalPublication No. WO2012/099805, each of which is herein incorporated byreference in its entirety).

The polymer may encapsulate the nanospecies or partially encapsulate thenanospecies. The immunogen may be a recombinant protein, a modified RNAand/or a polynucleotide described herein. In some embodiments, the lipidnanoparticle may be formulated for use in a vaccine such as, but notlimited to, against a pathogen.

Lipid nanoparticles may be engineered to alter the surface properties ofparticles so the lipid nanoparticles may penetrate the mucosal barrier.Mucus is located on mucosal tissue such as, but not limited to, oral(e.g., the buccal and esophageal membranes and tonsil tissue),ophthalmic, gastrointestinal (e.g., stomach, small intestine, largeintestine, colon, rectum), nasal, respiratory (e.g., nasal, pharyngeal,tracheal and bronchial membranes), genital (e.g., vaginal, cervical andurethral membranes). Nanoparticles larger than 10-200 nm which arepreferred for higher drug encapsulation efficiency and the ability toprovide the sustained delivery of a wide array of drugs have beenthought to be too large to rapidly diffuse through mucosal barriers.Mucus is continuously secreted, shed, discarded or digested and recycledso most of the trapped particles may be removed from the mucosal tissuewithin seconds or within a few hours. Large polymeric nanoparticles (200nm to 500 nm in diameter) which have been coated densely with a lowmolecular weight polyethylene glycol (PEG) diffused through mucus only 4to 6-fold lower than the same particles diffusing in water (Lai et al.PNAS 2007 104(5): 1482-487; Lai et al. Adv Drug Deliv Rev. 2009 61(2):158-171; each of which is herein incorporated by reference in itsentirety). The transport of nanoparticles may be determined using ratesof permeation and/or fluorescent microscopy techniques including, butnot limited to, fluorescence recovery after photobleaching (FRAP) andhigh resolution multiple particle tracking (MPT). As a non-limitingexample, compositions which can penetrate a mucosal barrier may be madeas described in U.S. Pat. No. 8,241,670 or International Publication No.WO2013/110028, the content of each of which is herein incorporated byreference in its entirety.

The lipid nanoparticle engineered to penetrate mucus may comprise apolymeric material (e.g., a polymeric core) and/or a polymer-vitaminconjugate and/or a tri-block co-polymer. The polymeric material mayinclude, but is not limited to, polyamines, polyethers, polyamides,polyesters, polycarbamates, polyureas, polycarbonates, poly(styrenes),polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes,polyethyeneimines, polyisocyanates, polyacrylates, polymethacrylates,polyacrylonitriles, and polyarylates. The polymeric material may bebiodegradable and/or biocompatible. Non-limiting examples ofbiocompatible polymers are described in International Publication No.WO2013/116804, the content of which is herein incorporated by referencein its entirety. The polymeric material may additionally be irradiated.As a non-limiting example, the polymeric material may be gammairradiated (see e.g., International Publication No. WO2012/082165,herein incorporated by reference in its entirety). Non-limiting examplesof specific polymers include poly(caprolactone) (PCL), ethylene vinylacetate polymer (EVA), poly(lactic acid) (PLA), poly(L-lactic acid)(PLLA), poly(glycolic acid) (PGA), poly(lactic acid-co-glycolic acid)(PLGA), poly(L-lactic acid-co-glycolic acid) (PLLGA), poly(D,L-lactide)(PDLA), poly(L-lactide) (PLLA), poly(D,L-lactide-co-caprolactone),poly(D,L-lactide-co-caprolactone-co-glycolide),poly(D,L-lactide-co-PEO-co-D,L-lactide),poly(D,L-lactide-co-PPO-co-D,L-lactide), polyalkyl cyanoacralate,polyurethane, poly-L-lysine (PLL), hydroxypropyl methacrylate (HPMA),polyethyleneglycol, poly-L-glutamic acid, poly(hydroxy acids),polyanhydrides, polyorthoesters, poly(ester amides), polyamides,poly(ester ethers), polycarbonates, polyalkylenes such as polyethyleneand polypropylene, polyalkylene glycols such as poly(ethylene glycol)(PEG), polyalkylene oxides (PEO), polyalkylene terephthalates such aspoly(ethylene terephthalate), polyvinyl alcohols (PVA), polyvinylethers, polyvinyl esters such as poly(vinyl acetate), polyvinyl halidessuch as poly(vinyl chloride) (PVC), polyvinylpyrrolidone, polysiloxanes,polystyrene (PS), polyurethanes, derivatized celluloses such as alkylcelluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters,nitro celluloses, hydroxypropylcellulose, carboxymethylcellulose,polymers of acrylic acids, such as poly(methyl(meth)acrylate) (PMMA),poly(ethyl(meth)acrylate), poly(butyl(meth)acrylate),poly(isobutyl(meth)acrylate), poly(hexyl(meth)acrylate),poly(isodecyl(meth)acrylate), poly(lauryl(meth)acrylate),poly(phenyl(meth)acrylate), poly(methyl acrylate), poly(isopropylacrylate), poly(isobutyl acrylate), poly(octadecyl acrylate) andcopolymers and mixtures thereof, polydioxanone and its copolymers,polyhydroxyalkanoates, polypropylene fumarate, polyoxymethylene,poloxamers, poly(ortho)esters, poly(butyric acid), poly(valeric acid),poly(lactide-co-caprolactone), PEG-PLGA-PEG and trimethylene carbonate,polyvinylpyrrolidone. The lipid nanoparticle may be coated or associatedwith a copolymer such as, but not limited to, a block co-polymer (suchas a branched polyether-polyamide block copolymer described inInternational Publication No. WO2013/012476, herein incorporated byreference in its entirety), and (poly(ethylene glycol))-(poly(propyleneoxide))-(poly(ethylene glycol)) triblock copolymer (see e.g., U.S.Publication 2012/0121718, U.S. Publication 2010/0003337 and U.S. Pat.No. 8,263,665, each of which is herein incorporated by reference in itsentirety). The co-polymer may be a polymer that is generally regarded assafe (GRAS) and the formation of the lipid nanoparticle may be in such away that no new chemical entities are created. For example, the lipidnanoparticle may comprise poloxamers coating PLGA nanoparticles withoutforming new chemical entities which are still able to rapidly penetratehuman mucus (Yang et al. Angew. Chem. Int. Ed. 2011 50:25972600, thecontent of which is herein incorporated by reference in its entirety). Anon-limiting scalable method to produce nanoparticles which canpenetrate human mucus is described by Xu et al. (see e.g., J ControlRelease 2013, 170(2):279-86, the content of which is herein incorporatedby reference in its entirety).

The vitamin of the polymer-vitamin conjugate may be vitamin E. Thevitamin portion of the conjugate may be substituted with other suitablecomponents such as, but not limited to, vitamin A, vitamin E, othervitamins, cholesterol, a hydrophobic moiety, or a hydrophobic componentof other surfactants (e.g., sterol chains, fatty acids, hydrocarbonchains and alkylene oxide chains).

In some embodiments, the RNA (e.g., mRNA) vaccine pharmaceuticalcompositions may be formulated in liposomes such as, but not limited to,DiLa2 liposomes (Marina Biotech, Bothell, Wash.), SMARTICLES® (MarinaBiotech, Bothell, Wash.), neutral DOPC(1,2-dioleoyl-sn-glycero-3-phosphocholine) based liposomes (e.g., siRNAdelivery for ovarian cancer (Landen et al. Cancer Biology & Therapy 20065(12)1708-1713, herein incorporated by reference in its entirety)) andhyaluronan-coated liposomes (Quiet Therapeutics, Israel).

In some embodiments, the RNA vaccines may be formulated in a lyophilizedgel-phase liposomal composition as described in U.S. Publication No.US2012/0060293, herein incorporated by reference in its entirety.

The nanoparticle formulations may comprise a phosphate conjugate. Thephosphate conjugate may increase in vivo circulation times and/orincrease the targeted delivery of the nanoparticle. Phosphate conjugatesfor use with the present invention may be made by the methods describedin International Publication No. WO2013/033438 or U.S. Publication No.2013/0196948, the content of each of which is herein incorporated byreference in its entirety. As a non-limiting example, the phosphateconjugates may include a compound of any one of the formulas describedin International Publication No. WO2013/033438, herein incorporated byreference in its entirety.

The nanoparticle formulation may comprise a polymer conjugate. Thepolymer conjugate may be a water soluble conjugate. The polymerconjugate may have a structure as described in U.S. Application No.2013/0059360, the content of which is herein incorporated by referencein its entirety. In some aspects, polymer conjugates with thepolynucleotides of the present invention may be made using the methodsand/or segmented polymeric reagents described in U.S. Patent ApplicationNo. 2013/0072709, herein incorporated by reference in its entirety. Inother aspects, the polymer conjugate may have pendant side groupscomprising ring moieties such as, but not limited to, the polymerconjugates described in U.S. Publication No. US2013/0196948, the contentof which is herein incorporated by reference in its entirety.

The nanoparticle formulations may comprise a conjugate to enhance thedelivery of nanoparticles of the present invention in a subject.Further, the conjugate may inhibit phagocytic clearance of thenanoparticles in a subject. In some aspects, the conjugate may be a“self” peptide designed from the human membrane protein CD47 (e.g., the“self” particles described by Rodriguez et al. (Science 2013, 339,971-975), herein incorporated by reference in its entirety). As shown byRodriguez et al. the self peptides delayed macrophage-mediated clearanceof nanoparticles which enhanced delivery of the nanoparticles. In otheraspects, the conjugate may be the membrane protein CD47 (e.g., seeRodriguez et al. Science 2013, 339, 971-975, herein incorporated byreference in its entirety). Rodriguez et al. showed that, similarly to“self” peptides, CD47 can increase the circulating particle ratio in asubject as compared to scrambled peptides and PEG coated nanoparticles.

In some embodiments, the RNA vaccines of the present invention areformulated in nanoparticles that comprise a conjugate to enhance thedelivery of the nanoparticles of the present disclosure in a subject.The conjugate may be the CD47 membrane or the conjugate may be derivedfrom the CD47 membrane protein, such as the “self” peptide describedpreviously. In other aspects the nanoparticle may comprise PEG and aconjugate of CD47 or a derivative thereof. In yet other aspects, thenanoparticle may comprise both the “self” peptide described above andthe membrane protein CD47.

In other aspects, a “self” peptide and/or CD47 protein may be conjugatedto a virus-like particle or pseudovirion, as described herein fordelivery of the RNA vaccines of the present invention.

In other embodiments, RNA vaccine pharmaceutical compositions comprisingthe polynucleotides of the present invention and a conjugate which mayhave a degradable linkage. Non-limiting examples of conjugates includean aromatic moiety comprising an ionizable hydrogen atom, a spacermoiety, and a water-soluble polymer. As a non-limiting example,pharmaceutical compositions comprising a conjugate with a degradablelinkage and methods for delivering such pharmaceutical compositions aredescribed in U.S. Publication No. US2013/0184443, the content of whichis herein incorporated by reference in its entirety.

The nanoparticle formulations may be a carbohydrate nanoparticlecomprising a carbohydrate carrier and a RNA (e.g., mRNA) vaccine. As anon-limiting example, the carbohydrate carrier may include, but is notlimited to, an anhydride-modified phytoglycogen or glycogen-typematerial, phytoglycogen octenyl succinate, phytoglycogen beta-dextrin,anhydride-modified phytoglycogen beta-dextrin. (See e.g., InternationalPublication No. WO2012/109121; the content of which is hereinincorporated by reference in its entirety).

Nanoparticle formulations of the present invention may be coated with asurfactant or polymer in order to improve the delivery of the particle.In some embodiments, the nanoparticle may be coated with a hydrophiliccoating such as, but not limited to, PEG coatings and/or coatings thathave a neutral surface charge. The hydrophilic coatings may help todeliver nanoparticles with larger payloads such as, but not limited to,RNA vaccines within the central nervous system. As a non-limitingexample nanoparticles comprising a hydrophilic coating and methods ofmaking such nanoparticles are described in U.S. Publication No.US2013/0183244, the content of which is herein incorporated by referencein its entirety.

In some embodiments, the lipid nanoparticles of the present inventionmay be hydrophilic polymer particles. Non-limiting examples ofhydrophilic polymer particles and methods of making hydrophilic polymerparticles are described in U.S. Publication No. US2013/0210991, thecontent of which is herein incorporated by reference in its entirety.

In other embodiments, the lipid nanoparticles of the present inventionmay be hydrophobic polymer particles.

Lipid nanoparticle formulations may be improved by replacing thecationic lipid with a biodegradable cationic lipid which is known as arapidly eliminated lipid nanoparticle (reLNP). Ionizable cationiclipids, such as, but not limited to, DLinDMA, DLin-KC2-DMA, andDLin-MC3-DMA, have been shown to accumulate in plasma and tissues overtime and may be a potential source of toxicity. The rapid metabolism ofthe rapidly eliminated lipids can improve the tolerability andtherapeutic index of the lipid nanoparticles by an order of magnitudefrom a 1 mg/kg dose to a 10 mg/kg dose in rat. Inclusion of anenzymatically degraded ester linkage can improve the degradation andmetabolism profile of the cationic component, while still maintainingthe activity of the reLNP formulation. The ester linkage can beinternally located within the lipid chain or it may be terminallylocated at the terminal end of the lipid chain. The internal esterlinkage may replace any carbon in the lipid chain.

In some embodiments, the internal ester linkage may be located on eitherside of the saturated carbo.

In some embodiments, an immune response may be elicited by delivering alipid nanoparticle which may include a nanospecies, a polymer and animmunogen (U.S. Publication No. 2012/0189700 and InternationalPublication No. WO2012/099805, each of which is herein incorporated byreference in its entirety).

The polymer may encapsulate the nanospecies or partially encapsulate thenanospecies. The immunogen may be a recombinant protein, a modified RNAand/or a polynucleotide described herein. In some embodiments, the lipidnanoparticle may be formulated for use in a vaccine such as, but notlimited to, against a pathogen.

Lipid nanoparticles may be engineered to alter the surface properties ofparticles so the lipid nanoparticles may penetrate the mucosal barrier.Mucus is located on mucosal tissue such as, but not limited to, oral(e.g., the buccal and esophageal membranes and tonsil tissue),ophthalmic, gastrointestinal (e.g., stomach, small intestine, largeintestine, colon, rectum), nasal, respiratory (e.g., nasal, pharyngeal,tracheal and bronchial membranes), genital (e.g., vaginal, cervical andurethral membranes). Nanoparticles larger than 10-200 nm which arepreferred for higher drug encapsulation efficiency and the ability toprovide the sustained delivery of a wide array of drugs have beenthought to be too large to rapidly diffuse through mucosal barriers.Mucus is continuously secreted, shed, discarded or digested and recycledso most of the trapped particles may be removed from the mucosal tissuewithin seconds or within a few hours. Large polymeric nanoparticles (200nm-500 nm in diameter) which have been coated densely with a lowmolecular weight polyethylene glycol (PEG) diffused through mucus only 4to 6-fold lower than the same particles diffusing in water (Lai et al.PNAS 2007 104(5): 1482-487; Lai et al. Adv Drug Deliv Rev. 2009 61(2):158-171; each of which is herein incorporated by reference in itsentirety). The transport of nanoparticles may be determined using ratesof permeation and/or fluorescent microscopy techniques including, butnot limited to, fluorescence recovery after photobleaching (FRAP) andhigh resolution multiple particle tracking (MPT). As a non-limitingexample, compositions which can penetrate a mucosal barrier may be madeas described in U.S. Pat. No. 8,241,670 or International Publication No.WO2013/110028, the content of each of which is herein incorporated byreference in its entirety.

The lipid nanoparticle engineered to penetrate mucus may comprise apolymeric material (i.e. a polymeric core) and/or a polymer-vitaminconjugate and/or a tri-block co-polymer. The polymeric material mayinclude, but is not limited to, polyamines, polyethers, polyamides,polyesters, polycarbamates, polyureas, polycarbonates, poly(styrenes),polyimides, polysulfones, polyurethanes, polyacetylenes, polyethylenes,polyethyeneimines, polyisocyanates, polyacrylates, polymethacrylates,polyacrylonitriles, and polyarylates. The polymeric material may bebiodegradable and/or biocompatible. Non-limiting examples ofbiocompatible polymers are described in International Publication No.WO2013/116804, the content of which is herein incorporated by referencein its entirety. The polymeric material may additionally be irradiated.As a non-limiting example, the polymeric material may be gammairradiated (see e.g., International Publication No. WO2012/082165,herein incorporated by reference in its entirety). Non-limiting examplesof specific polymers include poly(caprolactone) (PCL), ethylene vinylacetate polymer (EVA), poly(lactic acid) (PLA), poly(L-lactic acid)(PLLA), poly(glycolic acid) (PGA), poly(lactic acid-co-glycolic acid)(PLGA), poly(L-lactic acid-co-glycolic acid) (PLLGA), poly(D,L-lactide)(PDLA), poly(L-lactide) (PLLA), poly(D,L-lactide-co-caprolactone),poly(D,L-lactide-co-caprolactone-co-glycolide),poly(D,L-lactide-co-PEO-co-D,L-lactide),poly(D,L-lactide-co-PPO-co-D,L-lactide), polyalkyl cyanoacralate,polyurethane, poly-L-lysine (PLL), hydroxypropyl methacrylate (HPMA),polyethyleneglycol, poly-L-glutamic acid, poly(hydroxy acids),polyanhydrides, polyorthoesters, poly(ester amides), polyamides,poly(ester ethers), polycarbonates, polyalkylenes such as polyethyleneand polypropylene, polyalkylene glycols such as poly(ethylene glycol)(PEG), polyalkylene oxides (PEO), polyalkylene terephthalates such aspoly(ethylene terephthalate), polyvinyl alcohols (PVA), polyvinylethers, polyvinyl esters such as poly(vinyl acetate), polyvinyl halidessuch as poly(vinyl chloride) (PVC), polyvinylpyrrolidone, polysiloxanes,polystyrene (PS), polyurethanes, derivatized celluloses such as alkylcelluloses, hydroxyalkyl celluloses, cellulose ethers, cellulose esters,nitro celluloses, hydroxypropylcellulose, carboxymethylcellulose,polymers of acrylic acids, such as poly(methyl(meth)acrylate) (PMMA),poly(ethyl(meth)acrylate), poly(butyl(meth)acrylate),poly(isobutyl(meth)acrylate), poly(hexyl(meth)acrylate),poly(isodecyl(meth)acrylate), poly(lauryl(meth)acrylate),poly(phenyl(meth)acrylate), poly(methyl acrylate), poly(isopropylacrylate), poly(isobutyl acrylate), poly(octadecyl acrylate) andcopolymers and mixtures thereof, polydioxanone and its copolymers,polyhydroxyalkanoates, polypropylene fumarate, polyoxymethylene,poloxamers, poly(ortho)esters, poly(butyric acid), poly(valeric acid),poly(lactide-co-caprolactone), PEG-PLGA-PEG and trimethylene carbonate,polyvinylpyrrolidone. The lipid nanoparticle may be coated or associatedwith a copolymer such as, but not limited to, a block co-polymer (suchas a branched polyether-polyamide block copolymer described inInternational Publication No. WO2013/012476, herein incorporated byreference in its entirety), and (poly(ethylene glycol))-(poly(propyleneoxide))-(poly(ethylene glycol)) triblock copolymer (see e.g., U.S.Publication 2012/0121718 and U.S. Publication 2010/0003337 and U.S. Pat.No. 8,263,665; each of which is herein incorporated by reference in itsentirety). The co-polymer may be a polymer that is generally regarded assafe (GRAS) and the formation of the lipid nanoparticle may be in such away that no new chemical entities are created. For example, the lipidnanoparticle may comprise poloxamers coating PLGA nanoparticles withoutforming new chemical entities which are still able to rapidly penetratehuman mucus (Yang et al. Angew. Chem. Int. Ed. 2011 50:25972600; thecontent of which is herein incorporated by reference in its entirety). Anon-limiting scalable method to produce nanoparticles which canpenetrate human mucus is described by Xu et al. (see e.g., J ControlRelease 2013, 170(2):279-86, the content of which is herein incorporatedby reference in its entirety).

The vitamin of the polymer-vitamin conjugate may be vitamin E. Thevitamin portion of the conjugate may be substituted with other suitablecomponents such as, but not limited to, vitamin A, vitamin E, othervitamins, cholesterol, a hydrophobic moiety, or a hydrophobic componentof other surfactants (e.g., sterol chains, fatty acids, hydrocarbonchains and alkylene oxide chains).

The lipid nanoparticle engineered to penetrate mucus may include surfacealtering agents such as, but not limited to, polynucleotides, anionicproteins (e.g., bovine serum albumin), surfactants (e.g., cationicsurfactants such as for example dimethyldioctadecyl-ammonium bromide),sugars or sugar derivatives (e.g., cyclodextrin), nucleic acids,polymers (e.g., heparin, polyethylene glycol and poloxamer), mucolyticagents (e.g., N-acetylcysteine, mugwort, bromelain, papain,clerodendrum, acetylcysteine, bromhexine, carbocisteine, eprazinone,mesna, ambroxol, sobrerol, domiodol, letosteine, stepronin, tiopronin,gelsolin, thymosin β4 dornase alfa, neltenexine, erdosteine) and variousDNases including rhDNase.

The surface altering agent may be embedded or enmeshed in the particle'ssurface or disposed (e.g., by coating, adsorption, covalent linkage, orother process) on the surface of the lipid nanoparticle (see e.g., U.S.Publication 2010/0215580 and U.S. Publication 2008/0166414 andUS2013/0164343 the content of each of which is herein incorporated byreference in its entirety).

In some embodiments, the mucus penetrating lipid nanoparticles maycomprise at least one polynucleotide described herein. Thepolynucleotide may be encapsulated in the lipid nanoparticle and/ordisposed on the surface of the particle. The polynucleotide may becovalently coupled to the lipid nanoparticle. Formulations of mucuspenetrating lipid nanoparticles may comprise a plurality ofnanoparticles. Further, the formulations may contain particles which mayinteract with the mucus and alter the structural and/or adhesiveproperties of the surrounding mucus to decrease mucoadhesion which mayincrease the delivery of the mucus penetrating lipid nanoparticles tothe mucosal tissue.

In other embodiments, the mucus penetrating lipid nanoparticles may be ahypotonic formulation comprising a mucosal penetration enhancingcoating. The formulation may be hypotonic for the epithelium to which itis being delivered.

Non-limiting examples of hypotonic formulations may be found inInternational Publication No. WO2013/110028, the content of which isherein incorporated by reference in its entirety.

In some embodiments, in order to enhance the delivery through themucosal barrier the RNA vaccine formulation may comprise or be ahypotonic solution. Hypotonic solutions were found to increase the rateat which mucoinert particles such as, but not limited to,mucus-penetrating particles, were able to reach the vaginal epithelialsurface (see e.g., Ensign et al. Biomaterials 2013, 34(28):6922-9, thecontent of which is herein incorporated by reference in its entirety).

In some embodiments, the RNA vaccine is formulated as a lipoplex, suchas, without limitation, the ATUPLEX™ system, the DACC system, the DBTCsystem and other siRNA-lipoplex technology from Silence Therapeutics(London, United Kingdom), STEMFECT™ from STEMGENT® (Cambridge, Mass.),and polyethylenimine (PEI) or protamine-based targeted and non-targeteddelivery of nucleic acids (Aleku et al. Cancer Res. 2008 68:9788-9798;Strumberg et al. Int J Clin Pharmacol Ther 2012 50:76-78; Santel et al.,Gene Ther 2006 13:1222-1234; Santel et al., Gene Ther 2006 13:1360-1370;Gutbier et al., Pulm Pharmacol. Ther. 2010 23:334-344; Kaufmann et al.Microvasc Res 2010 80:286-293; Weide et al. J Immunother. 200932:498-507; Weide et al. J Immunother. 2008 31:180-188; Pascolo, ExpertOpin. Biol. Ther. 4:1285-1294; Fotin-Mleczek et al., 2011 J. Immunother.34:1-15; Song et al., Nature Biotechnol. 2005, 23:709-717; Peer et al.,Proc Natl Acad Sci USA. 2007 6; 104:4095-4100; deFougerolles Hum GeneTher. 2008 19:125-132; each of which is incorporated herein by referencein its entirety).

In some embodiments, such formulations may also be constructed orcompositions altered such that they passively or actively are directedto different cell types in vivo, including but not limited tohepatocytes, immune cells, tumor cells, endothelial cells, antigenpresenting cells, and leukocytes (Akinc et al. Mol Ther. 201018:1357-1364; Song et al., Nat Biotechnol. 2005 23:709-717; Judge etal., J Clin Invest. 2009 119:661-673; Kaufmann et al., Microvasc Res2010 80:286-293; Santel et al., Gene Ther 2006 13:1222-1234; Santel etal., Gene Ther 2006 13:1360-1370; Gutbier et al., Pulm Pharmacol. Ther.2010 23:334-344; Basha et al., Mol. Ther. 2011 19:2186-2200; Fenske andCullis, Expert Opin Drug Deliv. 2008 5:25-44; Peer et al., Science. 2008319:627-630; Peer and Lieberman, Gene Ther. 2011 18:1127-1133; each ofwhich is incorporated herein by reference in its entirety). One exampleof passive targeting of formulations to liver cells includes theDLin-DMA, DLin-KC2-DMA and DLin-MC3-DMA-based lipid nanoparticleformulations which have been shown to bind to apolipoprotein E andpromote binding and uptake of these formulations into hepatocytes invivo (Akinc et al. Mol Ther. 2010 18:1357-1364; herein incorporated byreference in its entirety). Formulations can also be selectivelytargeted through expression of different ligands on their surface asexemplified by, but not limited by, folate, transferrin,N-acetylgalactosamine (GalNAc), and antibody targeted approaches(Kolhatkar et al., Curr Drug Discov Technol. 2011 8:197-206; Musacchioand Torchilin, Front Biosci. 2011 16:1388-1412; Yu et al., MolMembrBiol. 2010 27:286-298; Patil et al., Crit Rev Ther Drug Carrier Syst.2008 25:1-61; Benoit et al., Biomacromolecules. 2011 12:2708-2714; Zhaoet al., Expert Opin Drug Deliv. 2008 5:309-319; Akinc et al., Mol Ther.2010 18:1357-1364; Srinivasan et al., Methods Mol Biol. 2012820:105-116; Ben-Arie et al., Methods Mol Biol. 2012 757:497-507; Peer2010 J Control Release. 20:63-68; Peer et al., Proc NatlAcad Sci USA.2007 104:4095-4100; Kim et al., Methods Mol Biol. 2011 721:339-353;Subramanya et al., Mol Ther. 2010 18:2028-2037; Song et al., NatBiotechnol. 2005 23:709-717; Peer et al., Science. 2008 319:627-630;Peer and Lieberman, Gene Ther. 2011 18:1127-1133; each of which isincorporated herein by reference in its entirety).

In some embodiments, the RNA (e.g., mRNA) vaccine is formulated as asolid lipid nanoparticle. A solid lipid nanoparticle (SLN) may bespherical with an average diameter between 10 to 1000 nm. SLN possess asolid lipid core matrix that can solubilize lipophilic molecules and maybe stabilized with surfactants and/or emulsifiers. In other embodiments,the lipid nanoparticle may be a self-assembly lipid-polymer nanoparticle(see Zhang et al., ACS Nano, 2008, 2 (8), pp 1696-1702; the content ofwhich is herein incorporated by reference in its entirety). As anon-limiting example, the SLN may be the SLN described in InternationalPublication No. WO2013/105101, the content of which is hereinincorporated by reference in its entirety. As another non-limitingexample, the SLN may be made by the methods or processes described inInternational Publication No. WO2013/105101, the content of which isherein incorporated by reference in its entirety.

Liposomes, lipoplexes, or lipid nanoparticles may be used to improve theefficacy of polynucleotides directed protein production as theseformulations may be able to increase cell transfection by the RNAvaccine; and/or increase the translation of encoded protein. One suchexample involves the use of lipid encapsulation to enable the effectivesystemic delivery of polyplex plasmid DNA (Heyes et al., Mol Ther. 200715:713-720; herein incorporated by reference in its entirety). Theliposomes, lipoplexes, or lipid nanoparticles may also be used toincrease the stability of the polynucleotide.

In some embodiments, the RNA (e.g., mRNA) vaccines of the presentinvention can be formulated for controlled release and/or targeteddelivery. As used herein, “controlled release” refers to apharmaceutical composition or compound release profile that conforms toa particular pattern of release to effect a therapeutic outcome. In someembodiments, the RNA vaccines may be encapsulated into a delivery agentdescribed herein and/or known in the art for controlled release and/ortargeted delivery. As used herein, the term “encapsulate” means toenclose, surround or encase. As it relates to the formulation of thecompounds of the invention, encapsulation may be substantial, completeor partial. The term “substantially encapsulated” means that at leastgreater than 50, 60, 70, 80, 85, 90, 95, 96, 97, 98, 99, 99.9, 99.9 orgreater than 99.999% of the pharmaceutical composition or compound ofthe invention may be enclosed, surrounded or encased within the deliveryagent. “Partially encapsulation” means that less than 10, 10, 20, 30, 4050 or less of the pharmaceutical composition or compound of theinvention may be enclosed, surrounded or encased within the deliveryagent. Advantageously, encapsulation may be determined by measuring theescape or the activity of the pharmaceutical composition or compound ofthe invention using fluorescence and/or electron micrograph. Forexample, at least 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 85, 90, 95, 96,97, 98, 99, 99.9, 99.99 or greater than 99.99% of the pharmaceuticalcomposition or compound of the present disclosure are encapsulated inthe delivery agent.

In some embodiments, the controlled release formulation may include, butis not limited to, tri-block co-polymers. As a non-limiting example, theformulation may include two different types of tri-block co-polymers(International Pub. No. WO2012/131104 and WO2012/131106; the contents ofeach of which is herein incorporated by reference in its entirety).

In other embodiments, the RNA vaccines may be encapsulated into a lipidnanoparticle or a rapidly eliminated lipid nanoparticle and the lipidnanoparticles or a rapidly eliminated lipid nanoparticle may then beencapsulated into a polymer, hydrogel and/or surgical sealant describedherein and/or known in the art. As a non-limiting example, the polymer,hydrogel or surgical sealant may be PLGA, ethylene vinyl acetate (EVAc),poloxamer, GELSITE® (Nanotherapeutics, Inc. Alachua, Fla.), HYLENEX®(Halozyme Therapeutics, San Diego Calif.), surgical sealants such asfibrinogen polymers (Ethicon Inc. Cornelia, Ga.), TISSELL® (BaxterInternational, Inc. Deerfield, Ill.), PEG-based sealants, and COSEAL®(Baxter International, Inc Deerfield, Ill.).

In other embodiments, the lipid nanoparticle may be encapsulated intoany polymer known in the art which may form a gel when injected into asubject. As another non-limiting example, the lipid nanoparticle may beencapsulated into a polymer matrix which may be biodegradable.

In some embodiments, the RNA vaccine formulation for controlled releaseand/or targeted delivery may also include at least one controlledrelease coating. Controlled release coatings include, but are notlimited to, OPADRY®, polyvinylpyrrolidone/vinyl acetate copolymer,polyvinylpyrrolidone, hydroxypropyl methylcellulose, hydroxypropylcellulose, hydroxyethyl cellulose, EUDRAGIT RL®, EUDRAGIT RS® andcellulose derivatives such as ethylcellulose aqueous dispersions(AQUACOAT® and SURELEASE®).

In some embodiments, the RNA (e.g., mRNA) vaccine controlled releaseand/or targeted delivery formulation may comprise at least onedegradable polyester which may contain polycationic side chains.Degradable polyesters include, but are not limited to, poly(serineester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester),and combinations thereof. In other embodiments, the degradablepolyesters may include a PEG conjugation to form a PEGylated polymer.

In some embodiments, the RNA vaccine controlled release and/or targeteddelivery formulation comprising at least one polynucleotide may compriseat least one PEG and/or PEG related polymer derivatives as described inU.S. Pat. No. 8,404,222, herein incorporated by reference in itsentirety.

In other embodiments, the RNA vaccine controlled release deliveryformulation comprising at least one polynucleotide may be the controlledrelease polymer system described in U.S. Publication No. 2013/0130348,herein incorporated by reference in its entirety.

In some embodiments, the RNA (e.g., mRNA)vaccines of the presentinvention may be encapsulated in a therapeutic nanoparticle, referred toherein as “therapeutic nanoparticle RNA vaccines.” Therapeuticnanoparticles may be formulated by methods described herein and known inthe art such as, but not limited to, International Publication Nos.WO2010/005740, WO2010/030763, WO2010/005721, WO2010/005723,WO2012/054923, U.S. Publication Nos. US2011/0262491, US2010/0104645,US2010/0087337, US2010/0068285, US2011/0274759, US2010/0068286,US2012/0288541, US2013/0123351 and US2013/0230567 and U.S. Pat. Nos.8,206,747, 8,293,276, 8,318,208 and 8,318,211, the content of each ofwhich is herein incorporated by reference in its entirety. In otherembodiments, therapeutic polymer nanoparticles may be identified by themethods described in U.S. Publication No. US2012/0140790, the content ofwhich is herein incorporated by reference in its entirety.

In some embodiments, the therapeutic nanoparticle RNA vaccine may beformulated for sustained release. As used herein, “sustained release”refers to a pharmaceutical composition or compound that conforms to arelease rate over a specific period of time. The period of time mayinclude, but is not limited to, hours, days, weeks, months and years. Asa non-limiting example, the sustained release nanoparticle may comprisea polymer and a therapeutic agent such as, but not limited to, thepolynucleotides of the present invention (see International PublicationNo. 2010/075072 and U.S. Publication Nos. US2010/0216804, US2011/0217377and US2012/0201859, each of which is herein incorporated by reference inits entirety). In another non-limiting example, the sustained releaseformulation may comprise agents which permit persistent bioavailabilitysuch as, but not limited to, crystals, macromolecular gels and/orparticulate suspensions (see U.S. Publication No. US2013/0150295, thecontent of which is herein incorporated by reference in its entirety).

In some embodiments, the therapeutic nanoparticle RNA vaccines may beformulated to be target specific. As a non-limiting example, thetherapeutic nanoparticles may include a corticosteroid (seeInternational Publication No. WO2011/084518, herein incorporated byreference in its entirety). As a non-limiting example, the therapeuticnanoparticles may be formulated in nanoparticles described inInternational Publication Nos. WO2008/121949, WO2010/005726,WO2010/005725, WO2011/084521 and U.S. Publication Nos. US2010/0069426,US2012/0004293 and US2010/0104655, each of which is herein incorporatedby reference in its entirety.

In some embodiments, the nanoparticles of the present invention maycomprise a polymeric matrix. As a non-limiting example, the nanoparticlemay comprise two or more polymers such as, but not limited to,polyethylenes, polycarbonates, polyanhydrides, polyhydroxyacids,polypropylfumerates, polycaprolactones, polyamides, polyacetals,polyethers, polyesters, poly(orthoesters), polycyanoacrylates, polyvinylalcohols, polyurethanes, polyphosphazenes, polyacrylates,polymethacrylates, polycyanoacrylates, polyureas, polystyrenes,polyamines, polylysine, poly(ethylene imine), poly(serine ester),poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester) orcombinations thereof.

In some embodiments, the therapeutic nanoparticle comprises a diblockcopolymer. In some embodiments, the diblock copolymer may include PEG incombination with a polymer such as, but not limited to, polyethylenes,polycarbonates, polyanhydrides, polyhydroxyacids, polypropylfumerates,polycaprolactones, polyamides, polyacetals, polyethers, polyesters,poly(orthoesters), polycyanoacrylates, polyvinyl alcohols,polyurethanes, polyphosphazenes, polyacrylates, polymethacrylates,polycyanoacrylates, polyureas, polystyrenes, polyamines, polylysine,poly(ethylene imine), poly(serine ester), poly(L-lactide-co-L-lysine),poly(4-hydroxy-L-proline ester) or combinations thereof. In yet otherembodiments, the diblock copolymer may be a high-X diblock copolymersuch as those described in International Publication No. WO2013/120052,the content of which is herein incorporated by reference in itsentirety.

As a non-limiting example, the therapeutic nanoparticle comprises aPLGA-PEG block copolymer (see U.S. Publication No. US2012/0004293 andU.S. Pat. No. 8,236,330, each of which is herein incorporated byreference in its entirety). In another non-limiting example, thetherapeutic nanoparticle is a stealth nanoparticle comprising a diblockcopolymer of PEG and PLA or PEG and PLGA (see U.S. Pat. No. 8,246,968and International Publication No. WO2012/166923, the content of each ofwhich is herein incorporated by reference in its entirety). In yetanother non-limiting example, the therapeutic nanoparticle is a stealthnanoparticle or a target-specific stealth nanoparticle as described inU.S. Publication No. 2013/0172406, the content of which is hereinincorporated by reference in its entirety.

In some embodiments, the therapeutic nanoparticle may comprise amultiblock copolymer (see e.g., U.S. Pat. Nos. 8,263,665 and 8,287,910and U.S. Publication No. 2013/0195987, the content of each of which isherein incorporated by reference in its entirety).

In yet another non-limiting example, the lipid nanoparticle comprisesthe block copolymer PEG-PLGA-PEG (see e.g., the thermosensitive hydrogel(PEG-PLGA-PEG) used as a TGF-beta1 gene delivery vehicle in Lee et al.“Thermosensitive Hydrogel as a TGF-β1 Gene Delivery Vehicle EnhancesDiabetic Wound Healing.” Pharmaceutical Research, 2003 20(12):1995-2000; and used as a controlled gene delivery system in Li et al.“Controlled Gene Delivery System Based on Thermosensitive BiodegradableHydrogel” Pharmaceutical Research 2003 20(6):884-888; and Chang et al.,“Non-ionic amphiphilic biodegradable PEG-PLGA-PEG copolymer enhancesgene delivery efficiency in rat skeletal muscle.” J Controlled Release.2007 118:245-253; each of which is herein incorporated by reference inits entirety). The RNA (e.g., mRNA) vaccines of the present disclosuremay be formulated in lipid nanoparticles comprising the PEG-PLGA-PEGblock copolymer.

In some embodiments, the block copolymers described herein may beincluded in a polyion complex comprising a non-polymeric micelle and theblock copolymer. (see e.g., U.S. Publication No. 2012/0076836, hereinincorporated by reference in its entirety).

In some embodiments, the therapeutic nanoparticle may comprise at leastone acrylic polymer. Acrylic polymers include but are not limited to,acrylic acid, methacrylic acid, acrylic acid and methacrylic acidcopolymers, methyl methacrylate copolymers, ethoxyethyl methacrylates,cyanoethyl methacrylate, amino alkyl methacrylate copolymer,poly(acrylic acid), poly(methacrylic acid), polycyanoacrylates andcombinations thereof.

In some embodiments, the therapeutic nanoparticles may comprise at leastone poly(vinyl ester) polymer. The poly(vinyl ester) polymer may be acopolymer such as a random copolymer. As a non-limiting example, therandom copolymer may have a structure such as those described inInternational Publication No. WO2013/032829 or U.S. Publication No.2013/0121954, the content of which is herein incorporated by referencein its entirety. In some aspects, the poly(vinyl ester) polymers may beconjugated to the polynucleotides described herein.

In some embodiments, the therapeutic nanoparticle may comprise at leastone diblock copolymer. The diblock copolymer may be, but it not limitedto, a poly(lactic) acid-poly(ethylene)glycol copolymer (see e.g.,International Publication No. WO2013/044219; herein incorporated byreference in its entirety). As a non-limiting example, the therapeuticnanoparticle may be used to treat cancer (see International publicationNo. WO2013/044219, herein incorporated by reference in its entirety).

In some embodiments, the therapeutic nanoparticles may comprise at leastone cationic polymer described herein and/or known in the art.

In some embodiments, the therapeutic nanoparticles may comprise at leastone amine-containing polymer such as, but not limited to polylysine,polyethyleneimine, poly(amidoamine) dendrimers, poly(beta-amino esters)(see e.g., U.S. Pat. No. 8,287,849, herein incorporated by reference inits entirety) and combinations thereof. In other embodiments, thenanoparticles described herein may comprise an amine cationic lipid suchas those described in International Publication No. WO2013/059496, thecontent of which is herein incorporated by reference in its entirety. Insome aspects the cationic lipids may have an amino-amine or anamino-amide moiety.

In some embodiments, the therapeutic nanoparticles may comprise at leastone degradable polyester, which may contain polycationic side chains.Degradable polyesters include, but are not limited to, poly(serineester), poly(L-lactide-co-L-lysine), poly(4-hydroxy-L-proline ester),and combinations thereof. In other embodiments, the degradablepolyesters may include a PEG conjugation to form a PEGylated polymer.

In other embodiments, the therapeutic nanoparticle may include aconjugation of at least one targeting ligand. The targeting ligand maybe any ligand known in the art such as, but not limited to, a monoclonalantibody (Kirpotin et al, Cancer Res. 2006 66:6732-6740, hereinincorporated by reference in its entirety).

In some embodiments, the therapeutic nanoparticle may be formulated inan aqueous solution, which may be used to target cancer (seeInternational Publication No. WO2011/084513 and U.S. Publication No.2011/0294717, each of which is herein incorporated by reference in itsentirety).

In some embodiments, the therapeutic nanoparticle RNA vaccines, e.g.,therapeutic nanoparticles comprising at least one RNA vaccine may beformulated using the methods described by Podobinski et al in U.S. Pat.No. 8,404,799, the content of which is herein incorporated by referencein its entirety.

In some embodiments, the RNA (e.g., mRNA) vaccines may be encapsulatedin, linked to and/or associated with synthetic nanocarriers. Syntheticnanocarriers include, but are not limited to, those described inInternational Publication Nos. WO2010/005740, WO2012/149454 andWO2013/019669, and U.S. Publication Nos. US2011/0262491, US2010/0104645,US2010/0087337 and US2012/0244222, each of which is herein incorporatedby reference in its entirety. The synthetic nanocarriers may beformulated using methods known in the art and/or described herein. As anon-limiting example, the synthetic nanocarriers may be formulated bythe methods described in International Publication Nos. WO2010/005740,WO2010/030763 and WO2012/13501, and U.S. Publication Nos.US2011/0262491, US2010/0104645, US2010/0087337 and US2012/024422, eachof which is herein incorporated by reference in its entirety. In otherembodiments, the synthetic nanocarrier formulations may be lyophilizedby methods described in International Publication No. WO2011/072218 andU.S. Pat. No. 8,211,473, the content of each of which is hereinincorporated by reference in its entirety. In yet other embodiments,formulations of the present invention, including, but not limited to,synthetic nanocarriers, may be lyophilized or reconstituted by themethods described in U.S. Publication No. 2013/0230568, the content ofwhich is herein incorporated by reference in its entirety.

In some embodiments, the synthetic nanocarriers may contain reactivegroups to release the polynucleotides described herein (seeInternational Publication No. WO2012/092552 and U.S. Publication No.US2012/0171229, each of which is herein incorporated by reference in itsentirety).

In some embodiments, the synthetic nanocarriers may contain animmunostimulatory agent to enhance the immune response from delivery ofthe synthetic nanocarrier. As a non-limiting example, the syntheticnanocarrier may comprise a Th1 immunostimulatory agent which may enhancea Th1-based response of the immune system (see International PublicationNo. WO2010/123569 and U.S. Publication No. 2011/0223201, each of whichis herein incorporated by reference in its entirety).

In some embodiments, the synthetic nanocarriers may be formulated fortargeted release. In some embodiments, the synthetic nanocarrier isformulated to release the polynucleotides at a specified pH and/or aftera desired time interval. As a non-limiting example, the syntheticnanoparticle may be formulated to release the RNA vaccines after 24hours and/or at a pH of 4.5 (see International Publication Nos.WO2010/138193 and WO2010/138194 and U.S. Publication Nos. US2011/0020388and US2011/0027217, each of which is herein incorporated by reference intheir entireties).

In some embodiments, the synthetic nanocarriers may be formulated forcontrolled and/or sustained release of the polynucleotides describedherein. As a non-limiting example, the synthetic nanocarriers forsustained release may be formulated by methods known in the art,described herein and/or as described in International Publication No.WO2010/138192 and U.S. Publication No. 2010/0303850, each of which isherein incorporated by reference in its entirety.

In some embodiments, the RNA vaccine may be formulated for controlledand/or sustained release wherein the formulation comprises at least onepolymer that is a crystalline side chain (CYSC) polymer. CYSC polymersare described in U.S. Pat. No. 8,399,007, herein incorporated byreference in its entirety.

In some embodiments, the synthetic nanocarrier may be formulated for useas a vaccine. In some embodiments, the synthetic nanocarrier mayencapsulate at least one polynucleotide which encode at least oneantigen. As a non-limiting example, the synthetic nanocarrier mayinclude at least one antigen and an excipient for a vaccine dosage form(see International Publication No. WO2011/150264 and U.S. PublicationNo. 2011/0293723, each of which is herein incorporated by reference inits entirety). As another non-limiting example, a vaccine dosage formmay include at least two synthetic nanocarriers with the same ordifferent antigens and an excipient (see International Publication No.WO2011/150249 and U.S. Publication No. 2011/0293701, each of which isherein incorporated by reference in its entirety). The vaccine dosageform may be selected by methods described herein, known in the artand/or described in International Publication No. WO2011/150258 and U.S.Publication No. US2012/0027806, each of which is herein incorporated byreference in its entirety).

In some embodiments, the synthetic nanocarrier may comprise at least onepolynucleotide which encodes at least one adjuvant. As non-limitingexample, the adjuvant may comprise dimethyldioctadecylammonium-bromide,dimethyldioctadecylammonium-chloride,dimethyldioctadecylammonium-phosphate ordimethyldioctadecylammonium-acetate (DDA) and an apolar fraction or partof said apolar fraction of a total lipid extract of a mycobacterium (seee.g., U.S. Pat. No. 8,241,610; herein incorporated by reference in itsentirety). In other embodiments, the synthetic nanocarrier may compriseat least one polynucleotide and an adjuvant. As a non-limiting example,the synthetic nanocarrier comprising and adjuvant may be formulated bythe methods described in International Publication No. WO2011/150240 andU.S. Publication No. US2011/0293700, each of which is hereinincorporated by reference in its entirety.

In some embodiments, the synthetic nanocarrier may encapsulate at leastone polynucleotide which encodes a peptide, fragment or region from avirus. As a non-limiting example, the synthetic nanocarrier may include,but is not limited to, the nanocarriers described in InternationalPublication Nos. WO2012/024621, WO2012/02629, WO2012/024632 and U.S.Publication No. US2012/0064110, US2012/0058153 and US2012/0058154, eachof which is herein incorporated by reference in its entirety.

In some embodiments, the synthetic nanocarrier may be coupled to apolynucleotide which may be able to trigger a humoral and/or cytotoxic Tlymphocyte (CTL) response (See e.g., International Publication No.WO2013/019669, herein incorporated by reference in its entirety).

In some embodiments, the RNA vaccine may be encapsulated in, linked toand/or associated with zwitterionic lipids. Non-limiting examples ofzwitterionic lipids and methods of using zwitterionic lipids aredescribed in U.S. Publication No. 2013/0216607, the content of which isherein incorporated by reference in its entirety. In some aspects, thezwitterionic lipids may be used in the liposomes and lipid nanoparticlesdescribed herein.

In some embodiments, the RNA vaccine may be formulated in colloidnanocarriers as described in U.S. Publication No. 2013/0197100, thecontent of which is herein incorporated by reference in its entirety.

In some embodiments, the nanoparticle may be optimized for oraladministration. The nanoparticle may comprise at least one cationicbiopolymer such as, but not limited to, chitosan or a derivativethereof. As a non-limiting example, the nanoparticle may be formulatedby the methods described in U.S. Publication No. 2012/0282343; hereinincorporated by reference in its entirety.

In some embodiments, LNPs comprise the lipid KL52 (an amino-lipiddisclosed in U.S. Application Publication No. 2012/0295832 expresslyincorporated herein by reference in its entirety). Activity and/orsafety (as measured by examining one or more of ALT/AST, white bloodcell count and cytokine induction) of LNP administration may be improvedby incorporation of such lipids. LNPs comprising KL52 may beadministered intravenously and/or in one or more doses. In someembodiments, administration of LNPs comprising KL52 results in equal orimproved mRNA and/or protein expression as compared to LNPs comprisingMC3.

In some embodiments, RNA vaccine may be delivered using smaller LNPs.Such particles may comprise a diameter from below 0.1 μm up to 100 nmsuch as, but not limited to, less than 0.1 μm, less than 1.0 μm, lessthan 5 μm, less than 10 μm, less than 15 μm, less than 20 μm, less than25 μm, less than 30 μm, less than 35 μm, less than 40 μm, less than 50jim, less than 55 μm, less than 60 μm, less than 65 μm, less than 70 μm,less than 75 μm, less than 80 μm, less than 85 μm, less than 90 μm, lessthan 95 μm, less than 100 μm, less than 125 μm, less than 150 μm, lessthan 175 μm, less than 200 μm, less than 225 μm, less than 250 μm, lessthan 275 μm, less than 300 μm, less than 325 μm, less than 350 μm, lessthan 375 μm, less than 400 μm, less than 425 μm, less than 450 μm, lessthan 475 μm, less than 500 μm, less than 525 μm, less than 550 μm, lessthan 575 μm, less than 600 μm, less than 625 μm, less than 650 μm, lessthan 675 μm, less than 700 μm, less than 725 μm, less than 750 μm, lessthan 775 μm, less than 800 μm, less than 825 μm, less than 850 μm, lessthan 875 μm, less than 900 μm, less than 925 μm, less than 950 μm, orless than 975 μm.

In other embodiments, RNA (e.g., mRNA) vaccines may be delivered usingsmaller LNPs which may comprise a diameter from about 1 nm to about 100nm, from about 1 nm to about 10 nm, about 1 nm to about 20 nm, fromabout 1 nm to about 30 nm, from about 1 nm to about 40 nm, from about 1nm to about 50 nm, from about 1 nm to about 60 nm, from about 1 nm toabout 70 nm, from about 1 nm to about 80 nm, from about 1 nm to about 90nm, from about 5 nm to about from 100 nm, from about 5 nm to about 10nm, about 5 nm to about 20 nm, from about 5 nm to about 30 nm, fromabout 5 nm to about 40 nm, from about 5 nm to about 50 nm, from about 5nm to about 60 nm, from about 5 nm to about 70 nm, from about 5 nm toabout 80 nm, from about 5 nm to about 90 nm, about 10 to about 50 nm,from about 20 to about 50 nm, from about 30 to about 50 nm, from about40 to about 50 nm, from about 20 to about 60 nm, from about 30 to about60 nm, from about 40 to about 60 nm, from about 20 to about 70 nm, fromabout 30 to about 70 nm, from about 40 to about 70 nm, from about 50 toabout 70 nm, from about 60 to about 70 nm, from about 20 to about 80 nm,from about 30 to about 80 nm, from about 40 to about 80 nm, from about50 to about 80 nm, from about 60 to about 80 nm, from about 20 to about90 nm, from about 30 to about 90 nm, from about 40 to about 90 nm, fromabout 50 to about 90 nm, from about 60 to about 90 nm and/or from about70 to about 90 nm.

In some embodiments, such LNPs are synthesized using methods comprisingmicrofluidic mixers. Exemplary microfluidic mixers may include, but arenot limited to a slit interdigital micromixer including, but not limitedto those manufactured by Microinnova (Allerheiligen bei Wildon, Austria)and/or a staggered herringbone micromixer (SHM) (Zhigaltsev, I. V. etal., Bottom-up design and synthesis of limit size lipid nanoparticlesystems with aqueous and triglyceride cores using millisecondmicrofluidic mixing have been published (Langmuir. 2012. 28:3633-40;Belliveau, N. M. et al., Microfluidic synthesis of highly potentlimit-size lipid nanoparticles for in vivo delivery of siRNA. MolecularTherapy-Nucleic Acids. 2012. 1:e37; Chen, D. et al., Rapid discovery ofpotent siRNA-containing lipid nanoparticles enabled by controlledmicrofluidic formulation. J Am Chem Soc. 2012. 134(16):6948-51; each ofwhich is herein incorporated by reference in its entirety).

In some embodiments, methods of LNP generation comprising SHM, furthercomprise the mixing of at least two input streams wherein mixing occursby microstructure-induced chaotic advection (MICA). According to thismethod, fluid streams flow through channels present in a herringbonepattern causing rotational flow and folding the fluids around eachother. This method may also comprise a surface for fluid mixing whereinthe surface changes orientations during fluid cycling. Methods ofgenerating LNPs using SHM include those disclosed in U.S. ApplicationPublication Nos. 2004/0262223 and 2012/0276209, each of which isexpressly incorporated herein by reference in their entirety.

In some embodiments, the RNA vaccine of the present invention may beformulated in lipid nanoparticles created using a micromixer such as,but not limited to, a Slit Interdigital Microstructured Mixer (SIMM-V2)or a Standard Slit Interdigital Micro Mixer (SSIMM) or Caterpillar(CPMM) or Impinging-j et (IJMM) from the Institut für Mikrotechnik MainzGmbH, Mainz Germany).

In some embodiments, the RNA (e.g., mRNA) vaccines of the presentdisclosure may be formulated in lipid nanoparticles created usingmicrofluidic technology (see Whitesides, George M. The Origins and theFuture of Microfluidics. Nature, 2006 442: 368-373; and Abraham et al.Chaotic Mixer for Microchannels. Science, 2002 295: 647-651; each ofwhich is herein incorporated by reference in its entirety). As anon-limiting example, controlled microfluidic formulation includes apassive method for mixing streams of steady pressure-driven flows inmicro channels at a low Reynolds number (see e.g., Abraham et al.Chaotic Mixer for Microchannels. Science, 2002 295: 647651; which isherein incorporated by reference in its entirety).

In some embodiments, the RNA (e.g., mRNA) vaccines of the presentinvention may be formulated in lipid nanoparticles created using amicromixer chip such as, but not limited to, those from HarvardApparatus (Holliston, Mass.) or Dolomite Microfluidics (Royston, UK). Amicromixer chip can be used for rapid mixing of two or more fluidstreams with a split and recombine mechanism.

In some embodiments, the RNA (e.g., mRNA) vaccines of the invention maybe formulated for delivery using the drug encapsulating microspheresdescribed in International Publication No. WO2013/063468 or U.S. Pat.No. 8,440,614, each of which is herein incorporated by reference in itsentirety. The microspheres may comprise a compound of the formula (I),(II), (III), (IV), (V) or (VI) as described in International PublicationNo. WO2013/063468, the content of which is herein incorporated byreference in its entirety. In other aspects, the amino acid, peptide,polypeptide, lipids (APPL) are useful in delivering the RNA vaccines ofthe invention to cells (see International Publication No. WO2013/063468,the contents of which is herein incorporated by reference in itsentirety).

In some embodiments, the RNA (e.g., mRNA) vaccines of the presentdisclosure may be formulated in lipid nanoparticles having a diameterfrom about 10 to about 100 nm such as, but not limited to, about 10 toabout 20 nm, about 10 to about 30 nm, about 10 to about 40 nm, about 10to about 50 nm, about 10 to about 60 nm, about 10 to about 70 nm, about10 to about 80 nm, about 10 to about 90 nm, about 20 to about 30 nm,about 20 to about 40 nm, about 20 to about 50 nm, about 20 to about 60nm, about 20 to about 70 nm, about 20 to about 80 nm, about 20 to about90 nm, about 20 to about 100 nm, about 30 to about 40 nm, about 30 toabout 50 nm, about 30 to about 60 nm, about 30 to about 70 nm, about 30to about 80 nm, about 30 to about 90 nm, about 30 to about 100 nm, about40 to about 50 nm, about 40 to about 60 nm, about 40 to about 70 nm,about 40 to about 80 nm, about 40 to about 90 nm, about 40 to about 100nm, about 50 to about 60 nm, about 50 to about 70 nm about 50 to about80 nm, about 50 to about 90 nm, about 50 to about 100 nm, about 60 toabout 70 nm, about 60 to about 80 nm, about 60 to about 90 nm, about 60to about 100 nm, about 70 to about 80 nm, about 70 to about 90 nm, about70 to about 100 nm, about 80 to about 90 nm, about 80 to about 100 nmand/or about 90 to about 100 nm.

In some embodiments, the lipid nanoparticles may have a diameter fromabout 10 to 500 nm.

In some embodiments, the lipid nanoparticle may have a diameter greaterthan 100 nm, greater than 150 nm, greater than 200 nm, greater than 250nm, greater than 300 nm, greater than 350 nm, greater than 400 nm,greater than 450 nm, greater than 500 nm, greater than 550 nm, greaterthan 600 nm, greater than 650 nm, greater than 700 nm, greater than 750nm, greater than 800 nm, greater than 850 nm, greater than 900 nm,greater than 950 nm or greater than 1000 nm.

In some aspects, the lipid nanoparticle may be a limit size lipidnanoparticle described in International Publication No. WO2013/059922,the content of which is herein incorporated by reference in itsentirety. The limit size lipid nanoparticle may comprise a lipid bilayersurrounding an aqueous core or a hydrophobic core; where the lipidbilayer may comprise a phospholipid such as, but not limited to,diacylphosphatidylcholine, a diacylphosphatidylethanolamine, a ceramide,a sphingomyelin, a dihydrosphingomyelin, a cephalin, a cerebroside, aC8-C20 fatty acid diacylphophatidylcholine, and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC). In other aspects the limit size lipidnanoparticle may comprise a polyethylene glycol-lipid such as, but notlimited to, DLPE-PEG, DMPE-PEG, DPPC-PEG and DSPE-PEG.

In some embodiments, the RNA vaccines may be delivered, localized and/orconcentrated in a specific location using the delivery methods describedin International Publication No. WO2013063530, the content of which isherein incorporated by reference in its entirety. As a non-limitingexample, a subject may be administered an empty polymeric particle priorto, simultaneously with or after delivering the RNA vaccines to thesubject. The empty polymeric particle undergoes a change in volume oncein contact with the subject and becomes lodged, embedded, immobilized orentrapped at a specific location in the subject.

In some embodiments, the RNA vaccines may be formulated in an activesubstance release system (see e.g., U.S. Publication No. US2013/0102545,the contents of which is herein incorporated by reference in itsentirety). The active substance release system may comprise 1) at leastone nanoparticle bonded to an oligonucleotide inhibitor strand which ishybridized with a catalytically active nucleic acid and 2) a compoundbonded to at least one substrate molecule bonded to a therapeuticallyactive substance (e.g., polynucleotides described herein), where thetherapeutically active substance is released by the cleavage of thesubstrate molecule by the catalytically active nucleic acid.

In some embodiments, the RNA (e.g., mRNA) vaccines may be formulated ina nanoparticle comprising an inner core comprising a non-cellularmaterial and an outer surface comprising a cellular membrane. Thecellular membrane may be derived from a cell or a membrane derived froma virus. As a non-limiting example, the nanoparticle may be made by themethods described in International Publication No. WO2013/052167, hereinincorporated by reference in its entirety. As another non-limitingexample, the nanoparticle described in International Publication No.WO2013/052167, herein incorporated by reference in its entirety, may beused to deliver the RNA vaccines described herein.

In some embodiments, the RNA vaccines may be formulated in porousnanoparticle-supported lipid bilayers (protocells). Protocells aredescribed in International Publication No. WO2013/056132, the content ofwhich is herein incorporated by reference in its entirety.

In some embodiments, the RNA vaccines described herein may be formulatedin polymeric nanoparticles as described in or made by the methodsdescribed in U.S. Pat. Nos. 8,420,123 and 8,518,963 and European PatentNo. EP2073848B1, the contents of each of which are herein incorporatedby reference in their entirety. As a non-limiting example, the polymericnanoparticle may have a high glass transition temperature such as thenanoparticles described in or nanoparticles made by the methodsdescribed in U.S. Pat. No. 8,518,963, the content of which is hereinincorporated by reference in its entirety. As another non-limitingexample, the polymer nanoparticle for oral and parenteral formulationsmay be made by the methods described in European Patent No. EP2073848B1,the content of which is herein incorporated by reference in itsentirety.

In other embodiments, the RNA (e.g., mRNA) vaccines described herein maybe formulated in nanoparticles used in imaging. The nanoparticles may beliposome nanoparticles such as those described in U.S. Publication No.2013/0129636, herein incorporated by reference in its entirety. As anon-limiting example, the liposome may comprisegadolinium(III)2-{4,7-bis-carboxymethyl-10-[(N,N-distearylamidomethyl-N′-amido-methyl]-1,4,7,10-tetra-azacyclododec-1-yl}-aceticacid and a neutral, fully saturated phospholipid component (see e.g.,U.S. Publication No US2013/0129636, the contents of which is hereinincorporated by reference in its entirety).

In some embodiments, the nanoparticles which may be used in the presentinvention are formed by the methods described in U.S. Patent ApplicationNo. 2013/0130348, the contents of which is herein incorporated byreference in its entirety.

The nanoparticles of the present invention may further include nutrientssuch as, but not limited to, those which deficiencies can lead to healthhazards from anemia to neural tube defects (see e.g., the nanoparticlesdescribed in International Patent Publication No. WO2013/072929, thecontents of which is herein incorporated by reference in its entirety).As a non-limiting example, the nutrient may be iron in the form offerrous, ferric salts or elemental iron, iodine, folic acid, vitamins ormicronutrients.

In some embodiments, the RNA (e.g., mRNA) vaccines of the presentinvention may be formulated in a swellable nanoparticle. The swellablenanoparticle may be, but is not limited to, those described in U.S. Pat.No. 8,440,231, the contents of which is herein incorporated by referencein its entirety. As a non-limiting embodiment, the swellablenanoparticle may be used for delivery of the RNA (e.g., mRNA) vaccinesof the present invention to the pulmonary system (see e.g., U.S. Pat.No. 8,440,231, the contents of which is herein incorporated by referencein its entirety).

The RNA (e.g., mRNA) vaccines of the present invention may be formulatedin polyanhydride nanoparticles such as, but not limited to, thosedescribed in U.S. Pat. No. 8,449,916, the contents of which is hereinincorporated by reference in its entirety. The nanoparticles andmicroparticles of the present invention may be geometrically engineeredto modulate macrophage and/or the immune response. In some aspects, thegeometrically engineered particles may have varied shapes, sizes and/orsurface charges in order to incorporated the polynucleotides of thepresent invention for targeted delivery such as, but not limited to,pulmonary delivery (see e.g., International Publication No.WO2013/082111, the content of which is herein incorporated by referencein its entirety). Other physical features the geometrically engineeringparticles may have include, but are not limited to, fenestrations,angled arms, asymmetry and surface roughness, charge which can alter theinteractions with cells and tissues. As a non-limiting example,nanoparticles of the present invention may be made by the methodsdescribed in International Publication No. WO2013/082111, the contentsof which is herein incorporated by reference in its entirety.

In some embodiments, the nanoparticles of the present invention may bewater soluble nanoparticles such as, but not limited to, those describedin International Publication No. WO2013/090601, the content of which isherein incorporated by reference in its entirety. The nanoparticles maybe inorganic nanoparticles which have a compact and zwitterionic ligandin order to exhibit good water solubility. The nanoparticles may alsohave small hydrodynamic diameters (HD), stability with respect to time,pH, and salinity and a low level of non-specific protein binding.

In some embodiments the nanoparticles of the present invention may bedeveloped by the methods described in U.S. Publication No.US2013/0172406, the content of which is herein incorporated by referencein its entirety.

In some embodiments, the nanoparticles of the present invention arestealth nanoparticles or target-specific stealth nanoparticles such as,but not limited to, those described in U.S. Publication No.2013/0172406, the content of which is herein incorporated by referencein its entirety. The nanoparticles of the present invention may be madeby the methods described in U.S. Publication No. 2013/0172406, thecontent of which is herein incorporated by reference in its entirety.

In other embodiments, the stealth or target-specific stealthnanoparticles may comprise a polymeric matrix. The polymeric matrix maycomprise two or more polymers such as, but not limited to,polyethylenes, polycarbonates, polyanhydrides, polyhydroxyacids,polypropylfumerates, polycaprolactones, polyamides, polyacetals,polyethers, polyesters, poly(orthoesters), polycyanoacrylates, polyvinylalcohols, polyurethanes, polyphosphazenes, polyacrylates,polymethacrylates, polycyanoacrylates, polyureas, polystyrenes,polyamines, polyesters, polyanhydrides, polyethers, polyurethanes,polymethacrylates, polyacrylates, polycyanoacrylates or combinationsthereof.

In some embodiments, the nanoparticle may be a nanoparticle-nucleic acidhybrid structure having a high density nucleic acid layer. As anon-limiting example, the nanoparticle-nucleic acid hybrid structure maymade by the methods described in U.S. Publication No. 2013/0171646, thecontent of which is herein incorporated by reference in its entirety.The nanoparticle may comprise a nucleic acid such as, but not limitedto, polynucleotides described herein and/or known in the art.

At least one of the nanoparticles of the present invention may beembedded in the core a nanostructure or coated with a low density porous3-D structure or coating which is capable of carrying or associatingwith at least one payload within or on the surface of the nanostructure.Non-limiting examples of the nanostructures comprising at least onenanoparticle are described in International Publication No.WO2013/123523, the content of which is herein incorporated by referencein its entirety.

In some embodiments, a nanoparticle comprises compounds of Formula (I):

or a salt or isomer thereof, wherein:

R₁ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀ alkenyl,—R*YR″, —YR″, and —R″M′R′;

R₂ and R₃ are independently selected from the group consisting of H,C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃,together with the atom to which they are attached, form a heterocycle orcarbocycle;

R₄ is selected from the group consisting of a C₃₋₆ carbocycle,—(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, —CQ(R)₂, and unsubstituted C₁₋₆alkyl, where Q is selected from a carbocycle, heterocycle, —OR,—O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H, —CXH₂, —CN, —N(R)₂,—C(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂,—N(R)R₈, —O(CH₂)_(n)OR, —N(R)C(═NR₉)N(R)₂, —N(R)C(═CHR₉)N(R)₂,—OC(O)N(R)₂, —N(R)C(O)OR, —N(OR)C(O)R, —N(OR)S(O)₂R, —N(OR)C(O)OR,—N(OR)C(O)N(R)₂, —N(OR)C(S)N(R)₂, —N(OR)C(═NR₉)N(R)₂,—N(OR)C(═CHR₉)N(R)₂, —C(═NR₉)N(R)₂, —C(═NR₉)R, —C(O)N(R)OR, and—C(R)N(R)₂C(O)OR, and each n is independently selected from 1, 2, 3, 4,and 5;

each R₅ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R₆ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, —S—S—, an aryl group, and a heteroaryl group;

R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;

R₈ is selected from the group consisting of C₃₋₆ carbocycle andheterocycle;

R₉ is selected from the group consisting of H, CN, NO₂, C₁₋₆ alkyl, —OR,—S(O)₂R, —S(O)₂N(R)₂, C₂₋₆ alkenyl, C₃₋₆ carbocycle and heterocycle;

each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R′ is independently selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;

each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl;

each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₂₋₁₂ alkenyl;

each Y is independently a C₃₋₆ carbocycle;

each X is independently selected from the group consisting of F, Cl, Br,and I; and

m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13.

In some embodiments, a subset of compounds of Formula (I) includes thosein which when R₄ is —(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, or —CQ(R)₂, then(i) Q is not —N(R)₂ when n is 1, 2, 3, 4 or 5, or (ii) Q is not 5, 6, or7-membered heterocycloalkyl when n is 1 or 2.

In some embodiments, another subset of compounds of Formula (I) includesthose in which

R₁ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀ alkenyl,—R*YR″, —YR″, and —R″M′R′;

R₂ and R₃ are independently selected from the group consisting of H,C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃,together with the atom to which they are attached, form a heterocycle orcarbocycle;

R₄ is selected from the group consisting of a C₃₋₆ carbocycle,—(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, —CQ(R)₂, and unsubstituted C₁₋₆alkyl, where Q is selected from a C₃₋₆ carbocycle, a 5- to 14-memberedheteroaryl having one or more heteroatoms selected from N, O, and S,—OR, —O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H, —CXH₂, —CN,—C(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂,—CRN(R)₂C(O)OR, —N(R)R₈, —O(CH₂)_(n)OR, —N(R)C(═NR₉)N(R)₂,—N(R)C(═CHR₉)N(R)₂, —OC(O)N(R)₂, —N(R)C(O)OR, —N(OR)C(O)R, —N(OR)S(O)₂R,—N(OR)C(O)OR, —N(OR)C(O)N(R)₂, —N(OR)C(S)N(R)₂, —N(OR)C(═NR₉)N(R)₂,—N(OR)C(═CHR₉)N(R)₂, —C(═NR₉)N(R)₂, —C(═NR₉)R, —C(O)N(R)OR, and a 5- to14-membered heterocycloalkyl having one or more heteroatoms selectedfrom N, O, and S which is substituted with one or more substituentsselected from oxo (═O), OH, amino, mono- or di-alkylamino, and C₁₋₃alkyl, and each n is independently selected from 1, 2, 3, 4, and 5;

each R₅ is independently selected from the group consisting of Ca-3alkyl, C₂₋₃ alkenyl, and H;

each R₆ is independently selected from the group consisting of Ca-3alkyl, C₂₋₃ alkenyl, and H;

M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, —S—S—, an aryl group, and a heteroaryl group;

R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;

R₈ is selected from the group consisting of C₃₋₆ carbocycle andheterocycle;

R₉ is selected from the group consisting of H, CN, NO₂, C₁₋₆ alkyl, —OR,—S(O)₂R, —S(O)₂N(R)₂, C₂₋₆ alkenyl, C₃₋₆ carbocycle and heterocycle;

each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R′ is independently selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;

each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl;

each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₂₋₁₂ alkenyl;

each Y is independently a C₃₋₆ carbocycle;

each X is independently selected from the group consisting of F, Cl, Br,and I; and

m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,

or salts or isomers thereof.

In some embodiments, another subset of compounds of Formula (I) includesthose in which

R₁ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀ alkenyl,—R*YR″, —YR″, and —R″M′R′;

R₂ and R₃ are independently selected from the group consisting of H,C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃,together with the atom to which they are attached, form a heterocycle orcarbocycle;

R₄ is selected from the group consisting of a C₃₋₆ carbocycle,—(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, —CQ(R)₂, and unsubstituted C₁₋₆alkyl, where Q is selected from a C₃₋₆ carbocycle, a 5- to 14-memberedheterocycle having one or more heteroatoms selected from N, O, and S,—OR, —O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H, —CXH₂, —CN,—C(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂,—CRN(R)₂C(O)OR, —N(R)R₈, —O(CH₂)_(n)OR, —N(R)C(═NR₉)N(R)₂,—N(R)C(═CHR₉)N(R)₂, —OC(O)N(R)₂, —N(R)C(O)OR, —N(OR)C(O)R, —N(OR)S(O)₂R,—N(OR)C(O)OR, —N(OR)C(O)N(R)₂, —N(OR)C(S)N(R)₂, —N(OR)C(═NR₉)N(R)₂,—N(OR)C(═CHR₉)N(R)₂, —C(═NR₉)R, —C(O)N(R)OR, and —C(═NR₉)N(R)₂, and eachn is independently selected from 1, 2, 3, 4, and 5; and when Q is a 5-to 14-membered heterocycle and (i) R₄ is —(CH₂)_(n)Q in which n is 1 or2, or (ii) R₄ is —(CH₂)_(n)CHQR in which n is 1, or (iii) R₄ is —CHQR,and —CQ(R)₂, then Q is either a 5- to 14-membered heteroaryl or 8- to14-membered heterocycloalkyl;

each R₅ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R₆ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, —S—S—, an aryl group, and a heteroaryl group;

R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;

R₈ is selected from the group consisting of C₃₋₆ carbocycle andheterocycle;

R₉ is selected from the group consisting of H, CN, NO₂, C₁₋₆ alkyl, —OR,—S(O)₂R, —S(O)₂N(R)₂, C₂₋₆ alkenyl, C₃₋₆ carbocycle and heterocycle;

each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R′ is independently selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;

each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl;

each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₂₋₁₂ alkenyl;

each Y is independently a C₃₋₆ carbocycle;

each X is independently selected from the group consisting of F, Cl, Br,and I; and

m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,

or salts or isomers thereof.

In some embodiments, another subset of compounds of Formula (I) includesthose in which

R₁ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀ alkenyl,—R*YR″, —YR″, and —R″M′R′;

R₂ and R₃ are independently selected from the group consisting of H,C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃,together with the atom to which they are attached, form a heterocycle orcarbocycle;

R₄ is selected from the group consisting of a C₃₋₆ carbocycle,—(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, —CQ(R)₂, and unsubstituted C₁₋₆alkyl, where Q is selected from a C₃₋₆ carbocycle, a 5- to 14-memberedheteroaryl having one or more heteroatoms selected from N, O, and S,—OR, —O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H, —CXH₂, —CN,—C(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂,—CRN(R)₂C(O)OR, —N(R)R₈, —O(CH₂)_(n)OR, —N(R)C(═NR₉)N(R)₂,—N(R)C(═CHR₉)N(R)₂, —OC(O)N(R)₂, —N(R)C(O)OR, —N(OR)C(O)R, —N(OR)S(O)₂R,—N(OR)C(O)OR, —N(OR)C(O)N(R)₂, —N(OR)C(S)N(R)₂, —N(OR)C(═NR₉)N(R)₂,—N(OR)C(═CHR₉)N(R)₂, —C(═NR₉)R, —C(O)N(R)OR, and —C(═NR₉)N(R)₂, and eachn is independently selected from 1, 2, 3, 4, and 5;

each R₅ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R₆ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, —S—S—, an aryl group, and a heteroaryl group; R₇ is selectedfrom the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl, and H;

R₈ is selected from the group consisting of C₃₋₆ carbocycle andheterocycle;

R₉ is selected from the group consisting of H, CN, NO₂, C₁₋₆ alkyl, —OR,—S(O)₂R, —S(O)₂N(R)₂, C₂₋₆ alkenyl, C₃₋₆ carbocycle and heterocycle;

each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R′ is independently selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;

each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl;

each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₂₋₁₂ alkenyl;

each Y is independently a C₃₋₆ carbocycle;

each X is independently selected from the group consisting of F, Cl, Br,and I; and

m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,

or salts or isomers thereof.

In some embodiments, another subset of compounds of Formula (I) includesthose in which

R₁ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀ alkenyl,—R*YR″, —YR″, and —R″M′R′;

R₂ and R₃ are independently selected from the group consisting of H,C₂₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃,together with the atom to which they are attached, form a heterocycle orcarbocycle;

R₄ is —(CH₂)_(n)Q or —(CH₂)_(n)CHQR, where Q is —N(R)₂, and n isselected from 3, 4, and 5;

each R₅ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R₆ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, —S—S—, an aryl group, and a heteroaryl group;

R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;

each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R′ is independently selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;

each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl;

each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₁₋₁₂ alkenyl;

each Y is independently a C₃₋₆ carbocycle;

each X is independently selected from the group consisting of F, Cl, Br,and I; and

m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,

or salts or isomers thereof.

In some embodiments, another subset of compounds of Formula (I) includesthose in which

R₁ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀ alkenyl,—R*YR″, —YR″, and —R″M′R′;

R₂ and R₃ are independently selected from the group consisting of C₁₋₁₄alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃, togetherwith the atom to which they are attached, form a heterocycle orcarbocycle;

R₄ is selected from the group consisting of —(CH₂)_(n)Q, —(CH₂)_(n)CHQR,—CHQR, and —CQ(R)₂, where Q is —N(R)₂, and n is selected from 1, 2, 3,4, and 5;

each R₅ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R₆ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, —S—S—, an aryl group, and a heteroaryl group;

R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;

each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;

each R′ is independently selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;

each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl;

each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₁₋₁₂ alkenyl;

each Y is independently a C₃₋₆ carbocycle;

each X is independently selected from the group consisting of F, Cl, Br,and I; and

m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,

or salts or isomers thereof.

In some embodiments, a subset of compounds of Formula (I) includes thoseof Formula (IA):

or a salt or isomer thereof, wherein l is selected from 1, 2, 3, 4, and5; m is selected from 5, 6, 7, 8, and 9; M₁ is a bond or M′; R₄ isunsubstituted C₁₋₃ alkyl, or —(CH₂)_(n)Q, in which Q is OH,—NHC(S)N(R)₂, —NHC(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)R₈,—NHC(═NR₉)N(R)₂, —NHC(═CHR₉)N(R)₂, —OC(O)N(R)₂, —N(R)C(O)OR, heteroarylor heterocycloalkyl; M and M′ are independently selected from —C(O)O—,—OC(O)—, —C(O)N(R′)—, —P(O)(OR′)O—, —S—S—, an aryl group, and aheteroaryl group; and R₂ and R₃ are independently selected from thegroup consisting of H, C₁₋₁₄ alkyl, and C₂₋₁₄ alkenyl.

In some embodiments, a subset of compounds of Formula (I) includes thoseof Formula (II):

or a salt or isomer thereof, wherein l is selected from 1, 2, 3, 4, and5; M₁ is a bond or M′; R₄ is unsubstituted C₁₋₃ alkyl, or —(CH₂)_(n)Q,in which n is 2, 3, or 4, and Q is OH, —NHC(S)N(R)₂, —NHC(O)N(R)₂,—N(R)C(O)R, —N(R)S(O)₂R, —N(R)R₈, —NHC(═NR₉)N(R)₂, —NHC(═CHR₉)N(R)₂,—OC(O)N(R)₂, —N(R)C(O)OR, heteroaryl or heterocycloalkyl; M and M′ areindependently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—, —P(O)(OR′)O—,—S—S—, an aryl group, and a heteroaryl group; and R₂ and R₃ areindependently selected from the group consisting of H, C₁₋₁₄ alkyl, andC₂₋₁₄ alkenyl.

In some embodiments, a subset of compounds of Formula (I) includes thoseof Formula (IIa), (IIb), (IIc), or (IIe):

or a salt or isomer thereof, wherein R₄ is as described herein.

In some embodiments, a subset of compounds of Formula (I) includes thoseof Formula (IId):

or a salt or isomer thereof, wherein n is 2, 3, or 4; and m, R′, R″, andR₂ through R₆ are as described herein. For example, each of R₂ and R₃may be independently selected from the group consisting of C₅₋₁₄ alkyland C₅₋₁₄ alkenyl.

In some embodiments, the compound of Formula (I) is selected from thegroup consisting of:

In further embodiments, the compound of Formula (I) is selected from thegroup consisting of:

In some embodiments, the compound of Formula (I) is selected from thegroup consisting of:

salts and isomers thereof.

In some embodiments, a nanoparticle comprises the following compound:

or salts and isomers thereof.

In some embodiments, the disclosure features a nanoparticle compositionincluding a lipid component comprising a compound as described herein(e.g., a compound according to Formula (I), (IA), (II), (IIa), (IIb),(IIc), (IId) or (IIe)).

In some embodiments, the disclosure features a pharmaceuticalcomposition comprising a nanoparticle composition according to thepreceding embodiments and a pharmaceutically acceptable carrier. Forexample, the pharmaceutical composition is refrigerated or frozen forstorage and/or shipment (e.g., being stored at a temperature of 4° C. orlower, such as a temperature between about −150° C. and about 0° C. orbetween about −80° C. and about −20° C. (e.g., about −5° C., −10° C.,−15° C., −20° C., −25° C., −30° C., −40° C., −50° C., −60° C., −70° C.,−80° C., −90° C., −130° C. or −150° C.). For example, the pharmaceuticalcomposition is a solution that is refrigerated for storage and/orshipment at, for example, about −20° C., −30° C., −40° C., −50° C., −60°C., −70° C., or −80° C.

In some embodiments, the disclosure provides a method of delivering atherapeutic and/or prophylactic (e.g., RNA, such as mRNA) to a cell(e.g., a mammalian cell). This method includes the step of administeringto a subject (e.g., a mammal, such as a human) a nanoparticlecomposition including (i) a lipid component including a phospholipid(such as a polyunsaturated lipid), a PEG lipid, a structural lipid, anda compound of Formula (I), (IA), (II), (IIa), (IIb), (IIc), (IId) or(IIe) and (ii) a therapeutic and/or prophylactic, in which administeringinvolves contacting the cell with the nanoparticle composition, wherebythe therapeutic and/or prophylactic is delivered to the cell.

In some embodiments, the disclosure provides a method of producing apolypeptide of interest in a cell (e.g., a mammalian cell). The methodincludes the step of contacting the cell with a nanoparticle compositionincluding (i) a lipid component including a phospholipid (such as apolyunsaturated lipid), a PEG lipid, a structural lipid, and a compoundof Formula (I), (IA), (II), (IIa), (IIb), (IIc), (IId) or (IIe) and (ii)an mRNA encoding the polypeptide of interest, whereby the mRNA iscapable of being translated in the cell to produce the polypeptide.

In some embodiments, the disclosure provides a method of treating adisease or disorder in a mammal (e.g., a human) in need thereof. Themethod includes the step of administering to the mammal atherapeutically effective amount of a nanoparticle composition including(i) a lipid component including a phospholipid (such as apolyunsaturated lipid), a PEG lipid, a structural lipid, and a compoundof Formula (I), (IA), (II), (IIa), (IIb), (IIc), (IId) or (IIe) and (ii)a therapeutic and/or prophylactic (e.g., an mRNA). In some embodiments,the disease or disorder is characterized by dysfunctional or aberrantprotein or polypeptide activity. For example, the disease or disorder isselected from the group consisting of rare diseases, infectiousdiseases, cancer and proliferative diseases, genetic diseases (e.g.,cystic fibrosis), autoimmune diseases, diabetes, neurodegenerativediseases, cardio- and reno-vascular diseases, and metabolic diseases.

In some embodiments, the disclosure provides a method of delivering(e.g., specifically delivering) a therapeutic and/or prophylactic to amammalian organ (e.g., a liver, spleen, lung, or femur). This methodincludes the step of administering to a subject (e.g., a mammal) ananoparticle composition including (i) a lipid component including aphospholipid, a PEG lipid, a structural lipid, and a compound of Formula(I), (IA), (II), (IIa), (IIb), (IIc), (IId) or (IIe) and (ii) atherapeutic and/or prophylactic (e.g., an mRNA), in which administeringinvolves contacting the cell with the nanoparticle composition, wherebythe therapeutic and/or prophylactic is delivered to the target organ(e.g., a liver, spleen, lung, or femur).

In some embodiments, the disclosure features a method for the enhanceddelivery of a therapeutic and/or prophylactic (e.g., an mRNA) to atarget tissue (e.g., a liver, spleen, lung, or femur). This methodincludes administering to a subject (e.g., a mammal) a nanoparticlecomposition, the composition including (i) a lipid component including acompound of Formula (I), (IA), (II), (IIa), (IIb), (IIc), (IId) or(IIe), a phospholipid, a structural lipid, and a PEG lipid; and (ii) atherapeutic and/or prophylactic, the administering including contactingthe target tissue with the nanoparticle composition, whereby thetherapeutic and/or prophylactic is delivered to the target tissue.

In some embodiments, the disclosure features a method of loweringimmunogenicity comprising introducing the nanoparticle composition ofthe disclosure into cells, wherein the nanoparticle composition reducesthe induction of the cellular immune response of the cells to thenanoparticle composition, as compared to the induction of the cellularimmune response in cells induced by a reference composition whichcomprises a reference lipid instead of a compound of Formula (I), (IA),(II), (IIa), (IIb), (IIc), (IId) or (IIe). For example, the cellularimmune response is an innate immune response, an adaptive immuneresponse, or both.

The disclosure also includes methods of synthesizing a compound ofFormula (I), (IA), (II), (IIa), (IIb), (IIc), (IId) or (IIe) and methodsof making a nanoparticle composition including a lipid componentcomprising the compound of Formula (I), (IA), (II), (IIa), (IIb), (IIc),(IId) or (IIe).

Modes of Vaccine Administration

VZV RNA (e.g., mRNA) vaccines may be administered by any route whichresults in a therapeutically effective outcome. These include, but arenot limited, to intradermal, intramuscular, intranasal, and/orsubcutaneous administration. The present disclosure provides methodscomprising administering RNA vaccines to a subject in need thereof. Theexact amount required will vary from subject to subject, depending onthe species, age, and general condition of the subject, the severity ofthe disease, the particular composition, its mode of administration, itsmode of activity, and the like. VZV RNA (e.g., mRNA) vaccinescompositions are typically formulated in dosage unit form for ease ofadministration and uniformity of dosage. It will be understood, however,that the total daily usage of VZV RNA (e.g., mRNA)vaccines compositionsmay be decided by the attending physician within the scope of soundmedical judgment. The specific therapeutically effective,prophylactically effective, or appropriate imaging dose level for anyparticular patient will depend upon a variety of factors including thedisorder being treated and the severity of the disorder; the activity ofthe specific compound employed; the specific composition employed; theage, body weight, general health, sex and diet of the patient; the timeof administration, route of administration, and rate of excretion of thespecific compound employed; the duration of the treatment; drugs used incombination or coincidental with the specific compound employed; andlike factors well known in the medical arts.

In some embodiments, VZV RNA (e.g., mRNA) vaccines compositions may beadministered at dosage levels sufficient to deliver 0.0001 mg/kg to 100mg/kg, 0.001 mg/kg to 0.05 mg/kg, 0.005 mg/kg to 0.05 mg/kg, 0.001 mg/kgto 0.005 mg/kg, 0.05 mg/kg to 0.5 mg/kg, 0.01 mg/kg to 50 mg/kg, 0.1mg/kg to 40 mg/kg, 0.5 mg/kg to 30 mg/kg, 0.01 mg/kg to 10 mg/kg, 0.1mg/kg to 10 mg/kg, or 1 mg/kg to 25 mg/kg, of subject body weight perday, one or more times a day, per week, per month, etc. to obtain thedesired therapeutic, diagnostic, prophylactic, or imaging effect (seee.g., the range of unit doses described in International Publication No.WO2013/078199, herein incorporated by reference in its entirety). Thedesired dosage may be delivered three times a day, two times a day, oncea day, every other day, every third day, every week, every two weeks,every three weeks, every four weeks, every 2 months, every three months,every 6 months, etc. In certain embodiments, the desired dosage may bedelivered using multiple administrations (e.g., two, three, four, five,six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen, ormore administrations). When multiple administrations are employed, splitdosing regimens such as those described herein may be used. In exemplaryembodiments, VZV RNA (e.g., mRNA) vaccines compositions may beadministered at dosage levels sufficient to deliver 0.0005 mg/kg to 0.01mg/kg, e.g., about 0.0005 mg/kg to about 0.0075 mg/kg, e.g., about0.0005 mg/kg, about 0.001 mg/kg, about 0.002 mg/kg, about 0.003 mg/kg,about 0.004 mg/kg or about 0.005 mg/kg.

In some embodiments, VZV RNA (e.g., mRNA) vaccine compositions may beadministered once or twice (or more) at dosage levels sufficient todeliver 0.025 mg/kg to 0.250 mg/kg, 0.025 mg/kg to 0.500 mg/kg, 0.025mg/kg to 0.750 mg/kg, or 0.025 mg/kg to 1.0 mg/kg.

In some embodiments, VZV RNA (e.g., mRNA) vaccine compositions may beadministered twice (e.g., Day 0 and Day 7, Day 0 and Day 14, Day 0 andDay 21, Day 0 and Day 28, Day 0 and Day 60, Day 0 and Day 90, Day 0 andDay 120, Day 0 and Day 150, Day 0 and Day 180, Day 0 and 3 months later,Day 0 and 6 months later, Day 0 and 9 months later, Day 0 and 12 monthslater, Day 0 and 18 months later, Day 0 and 2 years later, Day 0 and 5years later, or Day 0 and 10 years later) at a total dose of or atdosage levels sufficient to deliver a total dose of 0.0100 mg, 0.025 mg,0.050 mg, 0.075 mg, 0.100 mg, 0.125 mg, 0.150 mg, 0.175 mg, 0.200 mg,0.225 mg, 0.250 mg, 0.275 mg, 0.300 mg, 0.325 mg, 0.350 mg, 0.375 mg,0.400 mg, 0.425 mg, 0.450 mg, 0.475 mg, 0.500 mg, 0.525 mg, 0.550 mg,0.575 mg, 0.600 mg, 0.625 mg, 0.650 mg, 0.675 mg, 0.700 mg, 0.725 mg,0.750 mg, 0.775 mg, 0.800 mg, 0.825 mg, 0.850 mg, 0.875 mg, 0.900 mg,0.925 mg, 0.950 mg, 0.975 mg, or 1.0 mg. Higher and lower dosages andfrequency of administration are encompassed by the present disclosure.For example, a VZV RNA (e.g., mRNA) vaccine composition may beadministered three or four times.

In some embodiments, VZV RNA (e.g., mRNA) vaccine compositions may beadministered twice (e.g., Day 0 and Day 7, Day 0 and Day 14, Day 0 andDay 21, Day 0 and Day 28, Day 0 and Day 60, Day 0 and Day 90, Day 0 andDay 120, Day 0 and Day 150, Day 0 and Day 180, Day 0 and 3 months later,Day 0 and 6 months later, Day 0 and 9 months later, Day 0 and 12 monthslater, Day 0 and 18 months later, Day 0 and 2 years later, Day 0 and 5years later, or Day 0 and 10 years later) at a total dose of or atdosage levels sufficient to deliver a total dose of 0.010 mg, 0.025 mg,0.100 mg or 0.400 mg.

In some embodiments the VZV RNA (e.g., mRNA) vaccine for use in a methodof vaccinating a subject is administered the subject a single dosage ofbetween 10 μg/kg and 400 g/kg of the nucleic acid vaccine in aneffective amount to vaccinate the subject. In some embodiments the RNAvaccine for use in a method of vaccinating a subject is administered thesubject a single dosage of between 10 μg and 400 μg of the nucleic acidvaccine in an effective amount to vaccinate the subject. In someembodiments, a VZV RNA (e.g., mRNA) vaccine for use in a method ofvaccinating a subject is administered to the subject as a single dosageof 25-1000 μg (e.g., a single dosage of mRNA encoding an VZV antigen).In some embodiments, a VZV RNA vaccine is administered to the subject asa single dosage of 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500,550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000 μg. For example, aVZV RNA vaccine may be administered to a subject as a single dose of25-100, 25-500, 50-100, 50-500, 50-1000, 100-500, 100-1000, 250-500,250-1000, or 500-1000 μg. In some embodiments, a VZV RNA (e.g., mRNA)vaccine for use in a method of vaccinating a subject is administered tothe subject as two dosages, the combination of which equals 25-1000 μgof the VZV RNA (e.g., mRNA) vaccine.

A VZV RNA (e.g., mRNA) vaccine pharmaceutical composition describedherein can be formulated into a dosage form described herein, such as anintranasal, intratracheal, or injectable (e.g., intravenous,intraocular, intravitreal, intramuscular, intradermal, intracardiac,intraperitoneal, and subcutaneous).

VZV RNA vaccine formulations and methods of use Some aspects of thepresent disclosure provide formulations of the VZV RNA (e.g., mRNA)vaccine, wherein the VZV RNA vaccine is formulated in an effectiveamount to produce an antigen specific immune response in a subject(e.g., production of antibodies specific to an anti-VZV antigenicpolypeptide). “An effective amount” is a dose of an VZV RNA (e.g., mRNA)vaccine effective to produce an antigen-specific immune response. Alsoprovided herein are methods of inducing an antigen-specific immuneresponse in a subject.

In some embodiments, the antigen-specific immune response ischaracterized by measuring an anti-VZV antigenic polypeptide antibodytiter produced in a subject administered a VZV RNA (e.g., mRNA) vaccineas provided herein. An antibody titer is a measurement of the amount ofantibodies within a subject, for example, antibodies that are specificto a particular antigen (e.g., an anti-VZV antigenic polypeptide) orepitope of an antigen. Antibody titer is typically expressed as theinverse of the greatest dilution that provides a positive result.Enzyme-linked immunosorbent assay (ELISA) is a common assay fordetermining antibody titers, for example.

In some embodiments, an antibody titer is used to assess whether asubject has had an infection or to determine whether immunizations arerequired. In some embodiments, an antibody titer is used to determinethe strength of an autoimmune response, to determine whether a boosterimmunization is needed, to determine whether a previous vaccine waseffective, and to identify any recent or prior infections. In accordancewith the present disclosure, an antibody titer may be used to determinethe strength of an immune response induced in a subject by the VZV RNA(e.g., mRNA) vaccine.

In some embodiments, an anti-VZV antigenic polypeptide antibody titerproduced in a subject is increased by at least 1 log relative to acontrol. For example, anti-VZV antigenic polypeptide antibody titerproduced in a subject may be increased by at least 1.5, at least 2, atleast 2.5, or at least 3 log relative to a control. In some embodiments,the anti-VZV antigenic polypeptide antibody titer produced in thesubject is increased by 1, 1.5, 2, 2.5 or 3 log relative to a control.In some embodiments, the anti-VZV antigenic polypeptide antibody titerproduced in the subject is increased by 1-3 log relative to a control.For example, the anti-VZV antigenic polypeptide antibody titer producedin a subject may be increased by 1-1.5, 1-2, 1-2.5, 1-3, 1.5-2, 1.5-2.5,1.5-3, 2-2.5, 2-3, or 2.5-3 log relative to a control.

In some embodiments, the anti-VZV antigenic polypeptide antibody titerproduced in a subject is increased at least 2 times relative to acontrol. For example, the anti-VZV antigenic polypeptide antibody titerproduced in a subject may be increased at least 3 times, at least 4times, at least 5 times, at least 6 times, at least 7 times, at least 8times, at least 9 times, or at least 10 times relative to a control. Insome embodiments, the anti-VZV antigenic polypeptide antibody titerproduced in the subject is increased 2, 3, 4, 5, 6, 7, 8, 9, or 10 timesrelative to a control. In some embodiments, the anti-VZV antigenicpolypeptide antibody titer produced in a subject is increased 2-10 timesrelative to a control. For example, the anti-VZV antigenic polypeptideantibody titer produced in a subject may be increased 2-10, 2-9, 2-8,2-7, 2-6, 2-5, 2-4, 2-3, 3-10, 3-9, 3-8, 3-7, 3-6, 3-5, 3-4, 4-10, 4-9,4-8, 4-7, 4-6, 4-5, 5-10, 5-9, 5-8, 5-7, 5-6, 6-10, 6-9, 6-8, 6-7, 7-10,7-9, 7-8, 8-10, 8-9, or 9-10 times relative to a control.

A control, in some embodiments, is the anti-VZV antigenic polypeptideantibody titer produced in a subject who has not been administered a VZVRNA (e.g., mRNA) vaccine. In some embodiments, a control is an anti-VZVantigenic polypeptide antibody titer produced in a subject who has beenadministered a live attenuated VZV vaccine. An attenuated vaccine is avaccine produced by reducing the virulence of a viable (live). Anattenuated virus is altered in a manner that renders it harmless or lessvirulent relative to live, unmodified virus. In some embodiments, acontrol is an anti-VZV antigenic polypeptide antibody titer produced ina subject administered inactivated VZV vaccine. In some embodiments, acontrol is an anti-VZV antigenic polypeptide antibody titer produced ina subject administered a recombinant or purified VZV protein vaccine.Recombinant protein vaccines typically include protein antigens thateither have been produced in a heterologous expression system (e.g.,bacteria or yeast) or purified from large amounts of the pathogenicorganism. In some embodiments, a control is an anti-VZV antigenicpolypeptide antibody titer produced in a subject who has beenadministered a VZV virus-like particle (VLP) vaccine (e.g., particlesthat contain viral capsid protein but lack a viral genome and,therefore, cannot replicate/produce progeny virus). In some embodiments,the control is a VLP VZV vaccine that comprises prefusion or postfusionF proteins, or that comprises a combination of the two.

In some embodiments, an effective amount of a VZV RNA (e.g., mRNA)vaccine is a dose that is reduced compared to the standard of care doseof a recombinant VZV protein vaccine. A “standard of care,” as providedherein, refers to a medical or psychological treatment guideline and canbe general or specific. “Standard of care” specifies appropriatetreatment based on scientific evidence and collaboration between medicalprofessionals involved in the treatment of a given condition. It is thediagnostic and treatment process that a physician/clinician shouldfollow for a certain type of patient, illness or clinical circumstance.A “standard of care dose,” as provided herein, refers to the dose of arecombinant or purified VZV protein vaccine, or a live attenuated orinactivated VZV vaccine, or a VZV VLP vaccine, that aphysician/clinician or other medical professional would administer to asubject to treat or prevent VZV, or a VZV-related condition, whilefollowing the standard of care guideline for treating or preventing VZV,or a VZV-related condition.

In some embodiments, the anti-VZV antigenic polypeptide antibody titerproduced in a subject administered an effective amount of a VZV RNAvaccine is equivalent to an anti-VZV antigenic polypeptide antibodytiter produced in a control subject administered a standard of care doseof a recombinant or purified VZV protein vaccine, or a live attenuatedor inactivated VZV vaccine, or a VZV VLP vaccine.

In some embodiments, an effective amount of a VZV RNA (e.g., mRNA)vaccine is a dose equivalent to an at least 2-fold reduction in astandard of care dose of a recombinant or purified VZV protein vaccine.For example, an effective amount of a VZV RNA vaccine may be a doseequivalent to an at least 3-fold, at least 4-fold, at least 5-fold, atleast 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or atleast 10-fold reduction in a standard of care dose of a recombinant orpurified VZV protein vaccine. In some embodiments, an effective amountof a VZV RNA vaccine is a dose equivalent to an at least 100-fold, atleast 500-fold, or at least 1000-fold reduction in a standard of caredose of a recombinant or purified VZV protein vaccine. In someembodiments, an effective amount of a VZV RNA vaccine is a doseequivalent to a 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9-, 10-, 20-, 50-, 100-,250-, 500-, or 1000-fold reduction in a standard of care dose of arecombinant or purified VZV protein vaccine. In some embodiments, theanti-VZV antigenic polypeptide antibody titer produced in a subjectadministered an effective amount of a VZV RNA vaccine is equivalent toan anti-VZV antigenic polypeptide antibody titer produced in a controlsubject administered the standard of care dose of a recombinant orprotein VZV protein vaccine, or a live attenuated or inactivated VZVvaccine, or a VZV VLP vaccine. In some embodiments, an effective amountof a VZV RNA (e.g., mRNA) vaccine is a dose equivalent to a 2-fold to1000-fold (e.g., 2-fold to 100-fold, 10-fold to 1000-fold) reduction inthe standard of care dose of a recombinant or purified VZV proteinvaccine, wherein the anti-VZV antigenic polypeptide antibody titerproduced in the subject is equivalent to an anti-VZV antigenicpolypeptide antibody titer produced in a control subject administeredthe standard of care dose of a recombinant or purified VZV proteinvaccine, or a live attenuated or inactivated VZV vaccine, or a VZV VLPvaccine.

In some embodiments, the effective amount of a VZV RNA (e.g., mRNA)vaccine is a dose equivalent to a 2 to 1000-, 2 to 900-, 2 to 800-, 2 to700-, 2 to 600-, 2 to 500-, 2 to 400-, 2 to 300-, 2 to 200-, 2 to 100-,2 to 90-, 2 to 80-, 2 to 70-, 2 to 60-, 2 to 50-, 2 to 40-, 2 to 30-, 2to 20-, 2 to 10-, 2 to 9-, 2 to 8-, 2 to 7-, 2 to 6-, 2 to 5-, 2 to 4-,2 to 3-, 3 to 1000-, 3 to 900-, 3 to 800-, 3 to 700-, 3 to 600-, 3 to500-, 3 to 400-, 3 to 3 to 00-, 3 to 200-, 3 to 100-, 3 to 90-, 3 to80-, 3 to 70-, 3 to 60-, 3 to 50-, 3 to 40-, 3 to 30-, 3 to 20-, 3 to10-, 3 to 9-, 3 to 8-, 3 to 7-, 3 to 6-, 3 to 5-, 3 to 4-, 4 to 1000-, 4to 900-, 4 to 800-, 4 to 700-, 4 to 600-, 4 to 500-, 4 to 400-, 4 to300-, 4 to 200-, 4 to 100-, 4 to 90-, 4 to 80-, 4 to 70-, 4 to 60-, 4 to50-, 4 to 40-, 4 to 30-, 4 to 20-, 4 to 10-, 4 to 9-, 4 to 8-, 4 to 7-,4 to 6-, 4 to 5-, 4 to 4-, 5 to 1000-, 5 to 900-, 5 to 800-, 5 to 700-,5 to 600-, 5 to 500-, 5 to 400-, 5 to 300-, 5 to 200-, 5 to 100-, 5 to90-, 5 to 80-, 5 to 70-, 5 to 60-, 5 to 50-, 5 to 40-, 5 to 30-, 5 to20-, 5 to 10-, 5 to 9-, 5 to 8-S5 to 7-, 5 to 6-, 6 to 1000-, 6 to 900-,6 to 800-, 6 to 700-, 6 to 600-, 6 to 500-, 6 to 400-, 6 to 300-, 6 to200-, 6 to 100-, 6 to 90-, 6 to 80-, 6 to 70-, 6 to 60-, 6 to 50-, 6 to40-, 6 to 30-, 6 to 20-, 6 to 10-, 6 to 9-, 6 to 8-, 6 to 7-, 7 to1000-, 7 to 900-, 7 to 800-, 7 to 700-, 7 to 600-, 7 to 500-, 7 to 400-,7 to 300-, 7 to 200-, 7 to 100-, 7 to 90-, 7 to 80-, 7 to 70-, 7 to 60-,7 to 50-, 7 to 40-, 7 to 30-, 7 to 20-, 7 to 10-, 7 to 9-, 7 to 8-, 8 to1000-, 8 to 900-, 8 to 800-, 8 to 700-, 8 to 600-, 8 to 500-, 8 to 400-,8 to 300-, 8 to 200-, 8 to 100-, 8 to 90-, 8 to 80-, 8 to 70-, 8 to 60-,8 to 50-, 8 to 40-, 8 to 30-, 8 to 20-, 8 to 10-, 8 to 9-, 9 to 1000-, 9to 900-, 9 to 800-, 9 to 700-, 9 to 600-, 9 to 500-, 9 to 400-, 9 to300-, 9 to 200-, 9 to 100-, 9 to 90-, 9 to 80-, 9 to 70-, 9 to 60-, 9 to50-, 9 to 40-, 9 to 30-, 9 to 20-, 9 to 10-, 10 to 1000-, 10 to 900-, 10to 800-, 10 to 700-, 10 to 600-, 10 to 500-, 10 to 400-, 10 to 300-, 10to 200-, 10 to 100-, 10 to 90-, 10 to 80-, 10 to 70-, 10 to 60-, 10 to50-, 10 to 40-, 10 to 30-, 10 to 20-, 20 to 1000-, 20 to 900-, 20 to800-, 20 to 700-, 20 to 600-, 20 to 500-, 20 to 400-, 20 to 300-, 20 to200-, 20 to 100-, 20 to 90-, 20 to 80-, 20 to 70-, 20 to 60-, 20 to 50-,20 to 40-, 20 to 30-, 30 to 1000-, 30 to 900-, 30 to 800-, 30 to 700-,30 to 600-, 30 to 500-, 30 to 400-, 30 to 300-, 30 to 200-, 30 to 100-,30 to 90-, 30 to 80-, 30 to 70-, 30 to 60-, 30 to 50-, 30 to 40-, 40 to1000-, 40 to 900-, 40 to 800-, 40 to 700-, 40 to 600-, 40 to 500-, 40 to400-, 40 to 300-, 40 to 200-, 40 to 100-, 40 to 90-, 40 to 80-, 40 to70-, 40 to 60-, 40 to 50-, 50 to 1000-, 50 to 900-, 50 to 800-, 50 to700-, 50 to 600-, 50 to 500-, 50 to 400-, 50 to 300-, 50 to 200-, 50 to100-, 50 to 90-, 50 to 80-, 50 to 70-, 50 to 60-, 60 to 1000-, 60 to900-, 60 to 800-, 60 to 700-, 60 to 600-, 60 to 500-, 60 to 400-, 60 to300-, 60 to 200-, 60 to 100-, 60 to 90-, 60 to 80-, 60 to 70-, 70 to1000-, 70 to 900-, 70 to 800-, 70 to 700-, 70 to 600-, 70 to 500-, 70 to400-, 70 to 300-, 70 to 200-, 70 to 100-, 70 to 90-, 70 to 80-, 80 to1000-, 80 to 900-, 80 to 800-, 80 to 700-, 80 to 600-, 80 to 500-, 80 to400-, 80 to 300-, 80 to 200-, 80 to 100-, 80 to 90-, 90 to 1000-, 90 to900-, 90 to 800-, 90 to 700-, 90 to 600-, 90 to 500-, 90 to 400-, 90 to300-, 90 to 200-, 90 to 100-, 100 to 1000-, 100 to 900-, 100 to 800-,100 to 700-, 100 to 600-, 100 to 500-, 100 to 400-, 100 to 300-, 100 to200-, 200 to 1000-, 200 to 900-, 200 to 800-, 200 to 700-, 200 to 600-,200 to 500-, 200 to 400-, 200 to 300-, 300 to 1000-, 300 to 900-, 300 to800-, 300 to 700-, 300 to 600-, 300 to 500-, 300 to 400-, 400 to 1000-,400 to 900-, 400 to 800-, 400 to 700-, 400 to 600-, 400 to 500-, 500 to1000-, 500 to 900-, 500 to 800-, 500 to 700-, 500 to 600-, 600 to 1000-,600 to 900-, 600 to 800-, 600 to 700-, 700 to 1000-, 700 to 900-, 700 to800-, 800 to 1000-, 800 to 900-, or 900 to 1000-fold reduction in thestandard of care dose of a recombinant VZV protein vaccine. In someembodiments, such as the foregoing, the anti-VZV antigenic polypeptideantibody titer produced in the subject is equivalent to an anti-VZVantigenic polypeptide antibody titer produced in a control subjectadministered the standard of care dose of a recombinant or purified VZVprotein vaccine, or a live attenuated or inactivated VZV vaccine, or aVZV VLP vaccine. In some embodiments, the effective amount is a doseequivalent to (or equivalent to an at least) 2-, 3-, 4-, 5-, 6-, 7-, 8-,9-, 10-, 20-, 30-, 40-, 50-, 60-, 70-, 80-, 90-, 100-, 110-, 120-, 130-,140-, 150-, 160-, 170-, 1280-, 190-, 200-, 210-, 220-, 230-, 240-, 250-,260-, 270-, 280-, 290-, 300-, 310-, 320-, 330-, 340-, 350-, 360-, 370-,380-, 390-, 400-, 410-, 420-, 430-, 440-, 450-, 4360-, 470-, 480-, 490-,500-, 510-, 520-, 530-, 540-, 550-, 560-, 5760-, 580-, 590-, 600-, 610-,620-, 630-, 640-, 650-, 660-, 670-, 680-, 690-, 700-, 710-, 720-, 730-,740-, 750-, 760-, 770-, 780-, 790-, 800-, 810-, 820-, 830-, 840-, 850-,860-, 870-, 880-, 890-, 900-, 910-, 920-, 930-, 940-, 950-, 960-, 970-,980-, 990-, or 1000-fold reduction in the standard of care dose of arecombinant VZV protein vaccine. In some embodiments, such as theforegoing, an anti-VZV antigenic polypeptide antibody titer produced inthe subject is equivalent to an anti-VZV antigenic polypeptide antibodytiter produced in a control subject administered the standard of caredose of a recombinant or purified VZV protein vaccine, or a liveattenuated or inactivated VZV vaccine, or a VZV VLP vaccine.

In some embodiments, the effective amount of a VZV RNA (e.g., mRNA)vaccine is a total dose of 50-1000 μg. In some embodiments, theeffective amount of a VZV RNA (e.g., mRNA) vaccine is a total dose of50-1000, 50-900, 50-800, 50-700, 50-600, 50-500, 50-400, 50-300, 50-200,50-100, 50-90, 50-80, 50-70, 50-60, 60-1000, 60-900, 60-800, 60-700,60-600, 60-500, 60-400, 60-300, 60-200, 60-100, 60-90, 60-80, 60-70,70-1000, 70-900, 70-800, 70-700, 70-600, 70-500, 70-400, 70-300, 70-200,70-100, 70-90, 70-80, 80-1000, 80-900, 80-800, 80-700, 80-600, 80-500,80-400, 80-300, 80-200, 80-100, 80-90, 90-1000, 90-900, 90-800, 90-700,90-600, 90-500, 90-400, 90-300, 90-200, 90-100, 100-1000, 100-900,100-800, 100-700, 100-600, 100-500, 100-400, 100-300, 100-200, 200-1000,200-900, 200-800, 200-700, 200-600, 200-500, 200-400, 200-300, 300-1000,300-900, 300-800, 300-700, 300-600, 300-500, 300-400, 400-1000, 400-900,400-800, 400-700, 400-600, 400-500, 500-1000, 500-900, 500-800, 500-700,500-600, 600-1000, 600-900, 600-900, 600-700, 700-1000, 700-900,700-800, 800-1000, 800-900, or 900-1000 μg. In some embodiments, theeffective amount of a VZV RNA (e.g., mRNA) vaccine is a total dose of50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700,750, 800, 850, 900, 950 or 1000 μg. In some embodiments, the effectiveamount is a dose of 25-500 μg administered to the subject a total of twotimes. In some embodiments, the effective amount of a VZV RNA (e.g.,mRNA) vaccine is a dose of 25-500, 25-400, 25-300, 25-200, 25-100,25-50, 50-500, 50-400, 50-300, 50-200, 50-100, 100-500, 100-400,100-300, 100-200, 150-500, 150-400, 150-300, 150-200, 200-500, 200-400,200-300, 250-500, 250-400, 250-300, 300-500, 300-400, 350-500, 350-400,400-500 or 450-500 μg administered to the subject a total of two times.In some embodiments, the effective amount of a VZV RNA (e.g., mRNA)vaccine is a total dose of 25, 50, 100, 150, 200, 250, 300, 350, 400,450, or 500 μg administered to the subject a total of two times.

Additional Embodiments

1. A varicella zoster virus (VZV) vaccine, comprising:

at least one messenger ribonucleic acid (mRNA) polynucleotide having a5′ terminal cap, an open reading frame encoding at least one VZVantigenic polypeptide, and a 3′ polyA tail.

2. The vaccine of paragraph 1, wherein the at least one mRNApolynucleotide is encoded by a sequence identified by SEQ ID NO: 11.3. The vaccine of paragraph 1, wherein the at least one mRNApolynucleotide comprises a sequence identified by SEQ ID NO: 92.4. The vaccine of paragraph 1, wherein the at least one antigenicpolypeptide comprises a sequence identified by SEQ ID NO: 10.5. The vaccine of paragraph 1, wherein the at least one mRNApolynucleotide is encoded by a sequence identified by SEQ ID NO: 15.6. The vaccine of paragraph 1, wherein the at least one mRNApolynucleotide comprises a sequence identified by SEQ ID NO: 93.7. The vaccine of paragraph 1, wherein the at least one antigenicpolypeptide comprises a sequence identified by SEQ ID NO: 14.8. The vaccine of paragraph 1, wherein the at least one mRNApolynucleotide is encoded by a sequence identified by SEQ ID NO: 19.9. The vaccine of paragraph 1, wherein the at least one mRNApolynucleotide comprises a sequence identified by SEQ ID NO: 94.10. The vaccine of paragraph 1, wherein the at least one antigenicpolypeptide comprises a sequence identified by SEQ ID NO: 18.11. The vaccine of paragraph 1, wherein the at least one mRNApolynucleotide is encoded by a sequence identified by SEQ ID NO: 23.12. The vaccine of paragraph 1, wherein the at least one mRNApolynucleotide comprises a sequence identified by SEQ ID NO: 95.13. The vaccine of paragraph 1, wherein the at least one antigenicpolypeptide comprises a sequence identified by SEQ ID NO: 22.14. The vaccine of paragraph 1, wherein the at least one mRNApolynucleotide is encoded by a sequence identified by SEQ ID NO: 27.15. The vaccine of paragraph 1, wherein the at least one mRNApolynucleotide comprises a sequence identified by SEQ ID NO: 96.16. The vaccine of paragraph 1, wherein the at least one antigenicpolypeptide comprises a sequence identified by SEQ ID NO: 26.17. The vaccine of paragraph 1, wherein the at least one mRNApolynucleotide is encoded by a sequence identified by SEQ ID NO: 31.18. The vaccine of paragraph 1, wherein the at least one mRNApolynucleotide comprises a sequence identified by SEQ ID NO: 97.19. The vaccine of paragraph 1, wherein the at least one antigenicpolypeptide comprises a sequence identified by SEQ ID NO: 30.20. The vaccine of paragraph 1, wherein the at least one mRNApolynucleotide is encoded by a sequence identified by SEQ ID NO: 35.21. The vaccine of paragraph 1, wherein the at least one mRNApolynucleotide comprises a sequence identified by SEQ ID NO: 98.22. The vaccine of paragraph 1, wherein the at least one antigenicpolypeptide comprises a sequence identified by SEQ ID NO: 34.23. The vaccine of paragraph 1, wherein the at least one mRNApolynucleotide is encoded by a sequence identified by SEQ ID NO: 39.24. The vaccine of paragraph 1, wherein the at least one mRNApolynucleotide comprises a sequence identified by SEQ ID NO: 99.25. The vaccine of paragraph 1, wherein the at least one antigenicpolypeptide comprises a sequence identified by SEQ ID NO: 38.26. The vaccine of paragraph 1, wherein the at least one mRNApolynucleotide is encoded by a sequence identified by SEQ ID NO: 62.27. The vaccine of paragraph 1, wherein the at least one mRNApolynucleotide comprises a sequence identified by SEQ ID NO: 101.28. The vaccine of paragraph 26 or 27, wherein the at least oneantigenic polypeptide comprises a sequence identified by SEQ ID NO: 38.29. The vaccine of paragraph 1, wherein the at least one mRNApolynucleotide is encoded by a sequence identified by SEQ ID NO: 66.30. The vaccine of paragraph 1, wherein the at least one mRNApolynucleotide comprises a sequence identified by SEQ ID NO: 102.31. The vaccine of paragraph 30 or 31, wherein the at least oneantigenic polypeptide comprises a sequence identified by SEQ ID NO: 38.32. The vaccine of paragraph 1, wherein the at least one mRNApolynucleotide is encoded by a sequence identified by SEQ ID NO: 70.33. The vaccine of paragraph 1, wherein the at least one mRNApolynucleotide comprises a sequence identified by SEQ ID NO: 103.34. The vaccine of paragraph 32 or 33, wherein the at least oneantigenic polypeptide comprises a sequence identified by SEQ ID NO: 38.35. The vaccine of paragraph 1, wherein the at least one mRNApolynucleotide is encoded by a sequence identified by SEQ ID NO: 74.36. The vaccine of paragraph 1, wherein the at least one mRNApolynucleotide comprises a sequence identified by SEQ ID NO: 104.37. The vaccine of paragraph 35 or 36, wherein the at least oneantigenic polypeptide comprises a sequence identified by SEQ ID NO: 38.38. The vaccine of paragraph 1, wherein the at least one mRNApolynucleotide is encoded by a sequence identified by SEQ ID NO: 78.39. The vaccine of paragraph 1, wherein the at least one mRNApolynucleotide comprises a sequence identified by SEQ ID NO: 105.40. The vaccine of paragraph 38 or 39, wherein the at least oneantigenic polypeptide comprises a sequence identified by SEQ ID NO: 38.41. The vaccine of paragraph 1, wherein the at least one mRNApolynucleotide is encoded by a sequence identified by SEQ ID NO: 82.42. The vaccine of paragraph 1, wherein the at least one mRNApolynucleotide comprises a sequence identified by SEQ ID NO: 106.43. The vaccine of paragraph 41 or 42, wherein the at least oneantigenic polypeptide comprises a sequence identified by SEQ ID NO: 38.44. The vaccine of paragraph 1, wherein the at least one mRNApolynucleotide is encoded by a sequence identified by SEQ ID NO: 86.45. The vaccine of paragraph 1, wherein the at least one mRNApolynucleotide comprises a sequence identified by SEQ ID NO: 107, 134,or 148.46. The vaccine of paragraph 44 or 45, wherein the at least oneantigenic polypeptide comprises a sequence identified by SEQ ID NO: 38.47. The vaccine of paragraph 1, wherein the at least one mRNApolynucleotide is encoded by a sequence identified by SEQ ID NO: 90.48. The vaccine of paragraph 1, wherein the at least one mRNApolynucleotide comprises a sequence identified by SEQ ID NO: 108.49. The vaccine of paragraph 47 or 48, wherein the at least oneantigenic polypeptide comprises a sequence identified by SEQ ID NO: 38.50. The vaccine of any one of paragraphs 1-49, wherein the 5′ terminalcap is or comprises 7mG(5′)ppp(5′)NlmpNp.51. The vaccine of any one of paragraphs 1-50, wherein 100% of theuracil in the open reading frame is modified to include N1-methylpseudouridine at the 5-position of the uracil.52. The vaccine of any one of paragraphs 1-51, wherein the vaccine isformulated in a lipid nanoparticle comprising: DLin-MC3-DMA;cholesterol; 1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC); andpolyethylene glycol (PEG)2000-DMG.53. The vaccine of paragraph 52, wherein the lipid nanoparticle furthercomprises trisodium citrate buffer, sucrose and water.54. A varicella zoster virus (VZV) vaccine, comprising:

at least one messenger ribonucleic acid (mRNA) polynucleotide having a5′ terminal cap 7mG(5′)ppp(5′)NlmpNp, a sequence identified by SEQ IDNO: 92 and a 3′ polyA tail, wherein the uracil nucleotides of thesequence identified by SEQ ID NO: 92 are modified to include N1-methylpseudouridine at the 5-position of the uracil nucleotide.

55. A varicella zoster virus (VZV) vaccine, comprising:

at least one messenger ribonucleic acid (mRNA) polynucleotide having a5′ terminal cap 7mG(5′)ppp(5′)NlmpNp, a sequence identified by SEQ IDNO: 93 and a 3′ polyA tail, wherein the uracil nucleotides of thesequence identified by SEQ ID NO: 93 are modified to include N1-methylpseudouridine at the 5-position of the uracil nucleotide.

56. A varicella zoster virus (VZV) vaccine, comprising:

at least one messenger ribonucleic acid (mRNA) polynucleotide having a5′ terminal cap 7mG(5′)ppp(5′)NlmpNp, a sequence identified by SEQ IDNO: 94 and a 3′ polyA tail, wherein the uracil nucleotides of thesequence identified by SEQ ID NO: 94 are modified to include N1-methylpseudouridine at the 5-position of the uracil nucleotide.

57. A varicella zoster virus (VZV) vaccine, comprising:

at least one messenger ribonucleic acid (mRNA) polynucleotide having a5′ terminal cap 7mG(5′)ppp(5′)NlmpNp, a sequence identified by SEQ IDNO: 95 and a 3′ polyA tail, wherein the uracil nucleotides of thesequence identified by SEQ ID NO: 95 are modified to include N1-methylpseudouridine at the 5-position of the uracil nucleotide.

58. A varicella zoster virus (VZV) vaccine, comprising:

at least one messenger ribonucleic acid (mRNA) polynucleotide having a5′ terminal cap 7mG(5′)ppp(5′)NlmpNp, a sequence identified by SEQ IDNO: 96 and a 3′ polyA tail, wherein the uracil nucleotides of thesequence identified by SEQ ID NO: 96 are modified to include N1-methylpseudouridine at the 5-position of the uracil nucleotide.

59. A varicella zoster virus (VZV) vaccine, comprising:

at least one messenger ribonucleic acid (mRNA) polynucleotide having a5′ terminal cap 7mG(5′)ppp(5′)NlmpNp, a sequence identified by SEQ IDNO: 97 and a 3′ polyA tail, wherein the uracil nucleotides of thesequence identified by SEQ ID NO: 97 are modified to include N1-methylpseudouridine at the 5-position of the uracil nucleotide.

60. A varicella zoster virus (VZV) vaccine, comprising:

at least one messenger ribonucleic acid (mRNA) polynucleotide having a5′ terminal cap 7mG(5′)ppp(5′)NlmpNp, a sequence identified by SEQ IDNO: 98 and a 3′ polyA tail, wherein the uracil nucleotides of thesequence identified by SEQ ID NO: 98 are modified to include N1-methylpseudouridine at the 5-position of the uracil nucleotide.

61. A varicella zoster virus (VZV) vaccine, comprising:

at least one messenger ribonucleic acid (mRNA) polynucleotide having a5′ terminal cap 7mG(5′)ppp(5′)NlmpNp, a sequence identified by SEQ IDNO: 99 and a 3′ polyA tail, wherein the uracil nucleotides of thesequence identified by SEQ ID NO: 99 are modified to include N1-methylpseudouridine at the 5-position of the uracil nucleotide.

62. A varicella zoster virus (VZV) vaccine, comprising:

at least one messenger ribonucleic acid (mRNA) polynucleotide having a5′ terminal cap 7mG(5′)ppp(5′)NlmpNp, a sequence identified by SEQ IDNO: 101 and a 3′ polyA tail, wherein the uracil nucleotides of thesequence identified by SEQ ID NO: 101 are modified to include N1-methylpseudouridine at the 5-position of the uracil nucleotide.

63. A varicella zoster virus (VZV) vaccine, comprising:

at least one messenger ribonucleic acid (mRNA) polynucleotide having a5′ terminal cap 7mG(5′)ppp(5′)NlmpNp, a sequence identified by SEQ IDNO: 102 and a 3′ polyA tail, wherein the uracil nucleotides of thesequence identified by SEQ ID NO: 102 are modified to include N1-methylpseudouridine at the 5-position of the uracil nucleotide.

64. A varicella zoster virus (VZV) vaccine, comprising:

at least one messenger ribonucleic acid (mRNA) polynucleotide having a5′ terminal cap 7mG(5′)ppp(5′)NlmpNp, a sequence identified by SEQ IDNO: 103 and a 3′ polyA tail, wherein the uracil nucleotides of thesequence identified by SEQ ID NO: 103 are modified to include N1-methylpseudouridine at the 5-position of the uracil nucleotide.

65. A varicella zoster virus (VZV) vaccine, comprising:

at least one messenger ribonucleic acid (mRNA) polynucleotide having a5′ terminal cap 7mG(5′)ppp(5′)NlmpNp, a sequence identified by SEQ IDNO: 104 and a 3′ polyA tail, wherein the uracil nucleotides of thesequence identified by SEQ ID NO: 104 are modified to include N1-methylpseudouridine at the 5-position of the uracil nucleotide.

66. A varicella zoster virus (VZV) vaccine, comprising:

at least one messenger ribonucleic acid (mRNA) polynucleotide having a5′ terminal cap 7mG(5′)ppp(5′)NlmpNp, a sequence identified by SEQ IDNO: 105 and a 3′ polyA tail, wherein the uracil nucleotides of thesequence identified by SEQ ID NO: 105 are modified to include N1-methylpseudouridine at the 5-position of the uracil nucleotide.

67. A varicella zoster virus (VZV) vaccine, comprising:

at least one messenger ribonucleic acid (mRNA) polynucleotide having a5′ terminal cap 7mG(5′)ppp(5′)NlmpNp, a sequence identified by SEQ IDNO: 106 and a 3′ polyA tail, wherein the uracil nucleotides of thesequence identified by SEQ ID NO: 106 are modified to include N1-methylpseudouridine at the 5-position of the uracil nucleotide.

68. A varicella zoster virus (VZV) vaccine, comprising:

at least one messenger ribonucleic acid (mRNA) polynucleotide having a5′ terminal cap 7mG(5′)ppp(5′)NlmpNp, a sequence identified by SEQ IDNO: 107, 134, or 148 and a 3′ polyA tail, wherein the uracil nucleotidesof the sequence identified by SEQ ID NO: 107 or 134 are modified toinclude N1-methyl pseudouridine at the 5-position of the uracilnucleotide.

69. A varicella zoster virus (VZV) vaccine, comprising:

at least one messenger ribonucleic acid (mRNA) polynucleotide having a5′ terminal cap 7mG(5′)ppp(5′)NlmpNp, a sequence identified by SEQ IDNO: 108 and a 3′ polyA tail, wherein the uracil nucleotides of thesequence identified by SEQ ID NO: 108 are modified to include N1-methylpseudouridine at the 5-position of the uracil nucleotide.

70. The vaccine of any one of claims 54-69, wherein the vaccine isformulated in a lipid nanoparticle comprising DLin-MC3-DMA, cholesterol,1,2-Distearoyl-sn-glycero-3-phosphocholine (DSPC), and polyethyleneglycol (PEG)2000-DMG.71. The vaccine of any one of paragraphs 1-70 formulated in a lipidnanoparticle comprising at least one cationic lipid selected fromcompounds of Formula (I):

or a salt or isomer thereof, wherein:R₁ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀ alkenyl,—R*YR″, —YR″, and —R″M′R′;R₂ and R₃ are independently selected from the group consisting of H,C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃,together with the atom to which they are attached, form a heterocycle orcarbocycle;R₄ is selected from the group consisting of a C₃₋₆ carbocycle,—(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, —CQ(R)₂, and unsubstituted C₁₋₆alkyl, where Q is selected from a carbocycle, heterocycle, —OR,—O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H, —CXH₂, —CN, —N(R)₂,—C(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂,—N(R)R₈, —O(CH₂)_(n)OR, —N(R)C(═NR₉)N(R)₂, —N(R)C(═CHR₉)N(R)₂,—OC(O)N(R)₂, —N(R)C(O)OR, —N(OR)C(O)R, —N(OR)S(O)₂R, —N(OR)C(O)OR,—N(OR)C(O)N(R)₂, —N(OR)C(S)N(R)₂, —N(OR)C(═NR₉)N(R)₂,—N(OR)C(═CHR₉)N(R)₂, —C(═NR₉)N(R)₂, —C(═NR₉)R, —C(O)N(R)OR, and—C(R)N(R)₂C(O)OR, and each n is independently selected from 1, 2, 3, 4,and 5;each R₅ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;each R₆ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, —S—S—, an aryl group, and a heteroaryl group;R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;R₈ is selected from the group consisting of C₃₋₆ carbocycle andheterocycle;R₉ is selected from the group consisting of H, CN, NO₂, C₁₋₆ alkyl, —OR,—S(O)₂R, —S(O)₂N(R)₂, C₂₋₆ alkenyl, C₃₋₆ carbocycle and heterocycle;each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;each R′ is independently selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl;each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₂₋₁₂ alkenyl;each Y is independently a C₃₋₆ carbocycle;each X is independently selected from the group consisting of F, Cl, Br,and I; andm is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13.72. The vaccine of paragraph 71, wherein a subset of compounds ofFormula (I) includes those in which when R₄ is —(CH₂)_(n)Q,—(CH₂)_(n)CHQR, —CHQR, or —CQ(R)₂, then (i) Q is not —N(R)₂ when n is 1,2, 3, 4 or 5, or (ii) Q is not 5, 6, or 7-membered heterocycloalkyl whenn is 1 or 2.73. The vaccine of paragraph 71, wherein a subset of compounds ofFormula (I) includes those in whichR₁ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀ alkenyl,—R*YR″, —YR″, and —R″M′R′;R₂ and R₃ are independently selected from the group consisting of H,C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃,together with the atom to which they are attached, form a heterocycle orcarbocycle;R₄ is selected from the group consisting of a C₃₋₆ carbocycle,—(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, —CQ(R)₂, and unsubstituted C₁₋₆alkyl, where Q is selected from a C₃₋₆ carbocycle, a 5- to 14-memberedheteroaryl having one or more heteroatoms selected from N, O, and S,—OR, —O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H, —CXH₂, —CN,—C(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂,—CRN(R)₂C(O)OR, —N(R)R₈, —O(CH₂)_(n)OR, —N(R)C(═NR₉)N(R)₂,—N(R)C(═CHR₉)N(R)₂, —OC(O)N(R)₂, —N(R)C(O)OR, —N(OR)C(O)R, —N(OR)S(O)₂R,—N(OR)C(O)OR, —N(OR)C(O)N(R)₂, —N(OR)C(S)N(R)₂, —N(OR)C(═NR₉)N(R)₂,—N(OR)C(═CHR₉)N(R)₂, —C(═NR₉)N(R)₂, —C(═NR₉)R, —C(O)N(R)OR, and a 5- to14-membered heterocycloalkyl having one or more heteroatoms selectedfrom N, O, and S which is substituted with one or more substituentsselected from oxo (═O), OH, amino, mono- or di-alkylamino, and C₁₋₃alkyl, and each n is independently selected from 1, 2, 3, 4, and 5;each R₅ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;each R₆ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, —S—S—, an aryl group, and a heteroaryl group;R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;R₈ is selected from the group consisting of C₃₋₆ carbocycle andheterocycle;R₉ is selected from the group consisting of H, CN, NO₂, C₁₋₆ alkyl, —OR,—S(O)₂R, —S(O)₂N(R)₂, C₂₋₆ alkenyl, C₃₋₆ carbocycle and heterocycle;each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;each R′ is independently selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃0.1₄ alkenyl;each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₂₋₁₂ alkenyl;each Y is independently a C₃₋₆ carbocycle;each X is independently selected from the group consisting of F, Cl, Br,and I; andm is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,or salts or isomers thereof.74. The vaccine of paragraph 71, wherein a subset of compounds ofFormula (I) includes those in whichR₁ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀ alkenyl,—R*YR″, —YR″, and —R″M′R′;R₂ and R₃ are independently selected from the group consisting of H,C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃,together with the atom to which they are attached, form a heterocycle orcarbocycle;R₄ is selected from the group consisting of a C₃₋₆ carbocycle,—(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, —CQ(R)₂, and unsubstituted C₁₋₆alkyl, where Q is selected from a C₃₋₆ carbocycle, a 5- to 14-memberedheterocycle having one or more heteroatoms selected from N, O, and S,—OR, —O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H, —CXH₂, —CN,—C(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂,—CRN(R)₂C(O)OR, —N(R)R₈, —O(CH₂)_(n)OR, —N(R)C(═NR₉)N(R)₂,—N(R)C(═CHR₉)N(R)₂, —OC(O)N(R)₂, —N(R)C(O)OR, —N(OR)C(O)R, —N(OR)S(O)₂R,—N(OR)C(O)OR, —N(OR)C(O)N(R)₂, —N(OR)C(S)N(R)₂, —N(OR)C(═NR₉)N(R)₂,—N(OR)C(═CHR₉)N(R)₂, —C(═NR₉)R, —C(O)N(R)OR, and —C(═NR₉)N(R)₂, and eachn is independently selected from 1, 2, 3, 4, and 5; and when Q is a 5-to 14-membered heterocycle and (i) R₄ is —(CH₂)_(n)Q in which n is 1 or2, or (ii) R₄ is —(CH₂)_(n)CHQR in which n is 1, or (iii) R₄ is —CHQR,and —CQ(R)₂, then Q is either a 5- to 14-membered heteroaryl or 8- to14-membered heterocycloalkyl;each R₅ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;each R₆ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, —S—S—, an aryl group, and a heteroaryl group;R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;R₈ is selected from the group consisting of C₃₋₆ carbocycle andheterocycle;R₉ is selected from the group consisting of H, CN, NO₂, C₁₋₆ alkyl, —OR,—S(O)₂R, —S(O)₂N(R)₂, C₂₋₆ alkenyl, C₃₋₆ carbocycle and heterocycle;each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;each R′ is independently selected from the group consisting of C₁₋₈alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl;each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₂₋₁₂ alkenyl;each Y is independently a C₃₋₆ carbocycle;each X is independently selected from the group consisting of F, Cl, Br,and I; andm is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,or salts or isomers thereof.75. The vaccine of paragraph 71, wherein a subset of compounds ofFormula (I) includes those in whichR₁ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀ alkenyl,—R*YR″, —YR″, and —R″M′R′;R₂ and R₃ are independently selected from the group consisting of H,C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃,together with the atom to which they are attached, form a heterocycle orcarbocycle;R₄ is selected from the group consisting of a C₃₋₆ carbocycle,—(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, —CQ(R)₂, and unsubstituted C₁₋₆alkyl, where Q is selected from a C₃₋₆ carbocycle, a 5- to 14-memberedheteroaryl having one or more heteroatoms selected from N, O, and S,—OR, —O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H, —CXH₂, —CN,—C(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂,—CRN(R)₂C(O)OR, —N(R)R₈, —O(CH₂)_(n)OR, —N(R)C(═NR₉)N(R)₂,—N(R)C(═CHR₉)N(R)₂, —OC(O)N(R)₂, —N(R)C(O)OR, —N(OR)C(O)R, —N(OR)S(O)₂R,—N(OR)C(O)OR, —N(OR)C(O)N(R)₂, —N(OR)C(S)N(R)₂, —N(OR)C(═NR₉)N(R)₂,—N(OR)C(═CHR₉)N(R)₂, —C(═NR₉)R, —C(O)N(R)OR, and —C(═NR₉)N(R)₂, and eachn is independently selected from 1, 2, 3, 4, and 5;each R₅ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;each R₆ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, —S—S—, an aryl group, and a heteroaryl group;R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;R₈ is selected from the group consisting of C₃₋₆ carbocycle andheterocycle;R₉ is selected from the group consisting of H, CN, NO₂, C₁₋₆ alkyl, —OR,—S(O)₂R, —S(O)₂N(R)₂, C₂₋₆ alkenyl, C₃₋₆ carbocycle and heterocycle;each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;each R′ is independently selected from the group consisting of C₁₋₈alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl;each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₂₋₁₂ alkenyl;each Y is independently a C₃₋₆ carbocycle;each X is independently selected from the group consisting of F, Cl, Br,and I; andm is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,or salts or isomers thereof.76. The vaccine of paragraph 71, wherein a subset of compounds ofFormula (I) includes those in whichR₁ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀ alkenyl,—R*YR″, —YR″, and —R″M′R′;R₂ and R₃ are independently selected from the group consisting of H,C₂₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃,together with the atom to which they are attached, form a heterocycle orcarbocycle;R₄ is —(CH₂)_(n)Q or —(CH₂)_(n)CHQR, where Q is —N(R)₂, and n isselected from 3, 4, and 5;each R₅ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;each R₆ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, —S—S—, an aryl group, and a heteroaryl group;R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;each R′ is independently selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl;each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₁₋₁₂ alkenyl;each Y is independently a C₃₋₆ carbocycle;each X is independently selected from the group consisting of F, Cl, Br,and I; andm is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,or salts or isomers thereof.77. The vaccine of paragraph 71, wherein a subset of compounds ofFormula (I) includes those in whichR₁ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀ alkenyl,—R*YR″, —YR″, and —R″M′R′;R₂ and R₃ are independently selected from the group consisting of C₁₋₁₄alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃, togetherwith the atom to which they are attached, form a heterocycle orcarbocycle;R₄ is selected from the group consisting of —(CH₂)_(n)Q, —(CH₂)_(n)CHQR,—CHQR, and —CQ(R)₂, where Q is —N(R)₂, and n is selected from 1, 2, 3,4, and 5;each R₅ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;each R₆ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;M and M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, —S—S—, an aryl group, and a heteroaryl group;R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl,and H;each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H;each R′ is independently selected from the group consisting of C₁₋₁₈alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl;each R* is independently selected from the group consisting of C₁₋₁₂alkyl and C₁₋₁₂ alkenyl;each Y is independently a C₃₋₆ carbocycle;each X is independently selected from the group consisting of F, Cl, Br,and I; andm is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13,or salts or isomers thereof.78. The vaccine of paragraph 71, wherein a subset of compounds ofFormula (I) includes those of Formula (IA):

or a salt or isomer thereof, wherein l is selected from 1, 2, 3, 4, and5; m is selected from 5, 6, 7, 8, and 9; M₁ is a bond or M′; R₄ isunsubstituted C₁₋₃ alkyl, or —(CH₂)_(n)Q, in which Q is OH,—NHC(S)N(R)₂, —NHC(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)R₈,—NHC(═NR₉)N(R)₂, —NHC(═CHR₉)N(R)₂, —OC(O)N(R)₂, —N(R)C(O)OR, heteroarylor heterocycloalkyl; M and M′ are independently selected from —C(O)O—,—OC(O)—, —C(O)N(R′)—, —P(O)(OR′)O—, —S—S—, an aryl group, and aheteroaryl group; and R₂ and R₃ are independently selected from thegroup consisting of H, C₁₋₁₄ alkyl, and C₂₋₁₄ alkenyl.

This invention is not limited in its application to the details ofconstruction and the arrangement of components set forth in thefollowing description or illustrated in the drawings. The invention iscapable of other embodiments and of being practiced or of being carriedout in various ways. Also, the phraseology and terminology used hereinis for the purpose of description and should not be regarded aslimiting. The use of “including,” “comprising,” or “having,”“containing,” “involving,” and variations thereof herein, is meant toencompass the items listed thereafter and equivalents thereof as well asadditional items.

Broad Spectrum VZV Vaccines

It is envisioned that there may be situations where persons are at riskfor infection with more than one strain of VZV. RNA (e.g., mRNA)therapeutic vaccines are particularly amenable to combinationvaccination approaches due to a number of factors including, but notlimited to, speed of manufacture, ability to rapidly tailor vaccines toaccommodate perceived geographical threat, and the like. Moreover,because the vaccines utilize the human body to produce the antigenicprotein, the vaccines are amenable to the production of larger, morecomplex antigenic proteins, allowing for proper folding, surfaceexpression, antigen presentation, etc. in the human subject. To protectagainst more than one strain of VZV, a combination vaccine can beadministered that includes RNA encoding at least one antigenicpolypeptide protein (or antigenic portion thereof) of a first VZV andfurther includes RNA encoding at least one antigenic polypeptide protein(or antigenic portion thereof) of a second VZV. RNAs (mRNAs) can beco-formulated, for example, in a single lipid nanoparticle (LNP) or canbe formulated in separate LNPs destined for co-administration.

Multiprotein and Multicomponent Vaccines

The present disclosure encompasses VZV vaccines comprising multiple RNA(e.g., mRNA) polynucleotides, each encoding a single antigenicpolypeptide, as well as VZV vaccines comprising a single RNApolynucleotide encoding more than one antigenic polypeptide (e.g., as afusion polypeptide). Thus, it should be understood that a vaccinecomposition comprising a RNA (e.g., mRNA) polynucleotide having an openreading frame encoding a first VZV antigenic polypeptide and a RNApolynucleotide (e.g., mRNA) having an open reading frame encoding asecond VZV antigenic polypeptide encompasses (a) vaccines that comprisea first RNA polynucleotide encoding a first VZV antigenic polypeptideand a second RNA polynucleotide encoding a second VZV antigenicpolypeptide, and (b) vaccines that comprise a single RNA polynucleotideencoding a first and second VZV antigenic polypeptide (e.g., as a fusionpolypeptide). VZV RNA vaccines of the present disclosure, in someembodiments, comprise 2-10 (e.g., 2, 3, 4, 5, 6, 7, 8, 9 or 10), ormore, RNA polynucleotides having an open reading frame, each of whichencodes a different VZV antigenic polypeptide (or a single RNApolynucleotide encoding 2-10, or more, different VZV antigenicpolypeptides). In some embodiments, a VZV RNA vaccine comprises a RNApolynucleotide having an open reading frame encoding a VZV gE protein, aRNA polynucleotide having an open reading frame encoding a VZV gIprotein, a RNA polynucleotide having an open reading frame encoding aVZV gB protein, a RNA polynucleotide having an open reading frameencoding a VZV gH protein, a RNA polynucleotide having an open readingframe encoding a VZV gK protein, a RNA polynucleotide having an openreading frame encoding a VZV gL protein, a RNA polynucleotide having anopen reading frame encoding a VZV gC protein, a RNA polynucleotidehaving an open reading frame encoding a VZV gN protein, and a RNApolynucleotide having an open reading frame encoding a VZV gM protein.In some embodiments, a VZV RNA vaccine comprises a RNA polynucleotidehaving an open reading frame encoding a VZV gE and a RNA polynucleotidehaving an open reading frame encoding a VZV gI protein. In someembodiments, a VZV RNA vaccine comprises a RNA polynucleotide having anopen reading frame encoding a VZV gE protein or a gE variant.

In some embodiments, a RNA polynucleotide encodes a VZV antigenicpolypeptide fused to a signal peptide (e.g., SEQ ID NO: 56, 57, 109,110, or 111). The signal peptide may be fused at the N-terminus or theC-terminus of the antigenic polypeptide.

EXAMPLES Example 1: Manufacture of Polynucleotides

According to the present disclosure, the manufacture of polynucleotidesand/or parts or regions thereof may be accomplished utilizing themethods taught in International Publication WO2014/152027, entitled“Manufacturing Methods for Production of RNA Transcripts,” the contentsof which is incorporated herein by reference in its entirety.

Purification methods may include those taught in InternationalPublication WO2014/152030 and International Publication WO2014/152031,each of which is incorporated herein by reference in its entirety.

Detection and characterization methods of the polynucleotides may beperformed as taught in International Publication WO2014/144039, which isincorporated herein by reference in its entirety.

Characterization of the polynucleotides of the disclosure may beaccomplished using polynucleotide mapping, reverse transcriptasesequencing, charge distribution analysis, detection of RNA impurities,or any combination of two or more of the foregoing. “Characterizing”comprises determining the RNA transcript sequence, determining thepurity of the RNA transcript, or determining the charge heterogeneity ofthe RNA transcript, for example. Such methods are taught in, forexample, International Publication WO2014/144711 and InternationalPublication WO2014/144767, the content of each of which is incorporatedherein by reference in its entirety.

Example 2: Chimeric Polynucleotide Synthesis

According to the present disclosure, two regions or parts of a chimericpolynucleotide may be joined or ligated using triphosphate chemistry. Afirst region or part of 100 nucleotides or less is chemicallysynthesized with a 5′ monophosphate and terminal 3′desOH or blocked OH,for example. If the region is longer than 80 nucleotides, it may besynthesized as two strands for ligation.

If the first region or part is synthesized as a non-positionallymodified region or part using in vitro transcription (IVT), conversionthe 5′monophosphate with subsequent capping of the 3′ terminus mayfollow.

Monophosphate protecting groups may be selected from any of those knownin the art.

The second region or part of the chimeric polynucleotide may besynthesized using either chemical synthesis or IVT methods. IVT methodsmay include an RNA polymerase that can utilize a primer with a modifiedcap. Alternatively, a cap of up to 130 nucleotides may be chemicallysynthesized and coupled to the IVT region or part.

For ligation methods, ligation with DNA T4 ligase, followed by treatmentwith DNase should readily avoid concatenation.

The entire chimeric polynucleotide need not be manufactured with aphosphate-sugar backbone. If one of the regions or parts encodes apolypeptide, then such region or part may comprise a phosphate-sugarbackbone.

Ligation is then performed using any known click chemistry, orthoclickchemistry, solulink, or other bioconjugate chemistries known to those inthe art.

Synthetic Route

The chimeric polynucleotide may be made using a series of startingsegments. Such segments include:

(a) a capped and protected 5′ segment comprising a normal 3′OH (SEG. 1)

(b) a 5′ triphosphate segment, which may include the coding region of apolypeptide and a normal 3′OH (SEG. 2)

(c) a 5′ monophosphate segment for the 3′ end of the chimericpolynucleotide (e.g., the tail) comprising cordycepin or no 3′OH (SEG.3)

After synthesis (chemical or IVT), segment 3 (SEG. 3) may be treatedwith cordycepin and then with pyrophosphatase to create the 5′monophosphate.

Segment 2 (SEG. 2) may then be ligated to SEG. 3 using RNA ligase. Theligated polynucleotide is then purified and treated with pyrophosphataseto cleave the diphosphate. The treated SEG.2-SEG. 3 construct may thenbe purified and SEG. 1 is ligated to the 5′ terminus. A furtherpurification step of the chimeric polynucleotide may be performed.

Where the chimeric polynucleotide encodes a polypeptide, the ligated orjoined segments may be represented as: 5′UTR (SEG. 1), open readingframe or ORF (SEG. 2) and 3′UTR+PolyA (SEG. 3).

The yields of each step may be as much as 90-95%.

Example 3: PCR for cDNA Production

PCR procedures for the preparation of cDNA may be performed using 2×KAPA HIFI™ HotStart ReadyMix by Kapa Biosystems (Woburn, Mass.). Thissystem includes 2× KAPA ReadyMix 12.5 μl; Forward Primer (10 μM) 0.75μl; Reverse Primer (10 μM) 0.75 μl; Template cDNA 100 ng; and dH₂0diluted to 25.0 μl. The reaction conditions may be at 95° C. for 5 min.The reaction may be performed for 25 cycles of 98° C. for 20 sec, then58° C. for 15 sec, then 72° C. for 45 sec, then 72° C. for 5 min, then4° C. to termination.

The reaction may be cleaned up using Invitrogen's PURELINK™ PCR MicroKit (Carlsbad, Calif.) per manufacturer's instructions (up to 5 μg).Larger reactions may require a cleanup using a product with a largercapacity. Following the cleanup, the cDNA may be quantified using theNANODROP™ and analyzed by agarose gel electrophoresis to confirm thatthe cDNA is the expected size. The cDNA may then be submitted forsequencing analysis before proceeding to the in vitro transcriptionreaction.

Example 4: In Vitro Transcription (IVT)

The in vitro transcription reaction generates RNA polynucleotides. Suchpolynucleotides may comprise a region or part of the polynucleotides ofthe disclosure, including chemically modified RNA (e.g., mRNA)polynucleotides. The chemically modified RNA polynucleotides can beuniformly modified polynucleotides. The in vitro transcription reactionutilizes a custom mix of nucleotide triphosphates (NTPs). The NTPs maycomprise chemically modified NTPs, or a mix of natural and chemicallymodified NTPs, or natural NTPs.

A typical in vitro transcription reaction includes the following:

1) Template cDNA 1.0 μg 2) 10x transcription buffer 2.0 μl (400 mMTris-HCl pH 8.0, 190 mM MgCl₂, 50 mM DTT, 10 mM Spermidine) 3) CustomNTPs (25 mM each) 0.2 μl 4) RNase Inhibitor 20 U 5) T7 RNA polymerase3000 U 6) dH₂0 up to 20.0 μl. and 7) Incubation at 37° C. for 3 hr-5hrs.

The crude IVT mix may be stored at 4° C. overnight for cleanup the nextday. 1 U of RNase-free DNase may then be used to digest the originaltemplate. After 15 minutes of incubation at 37° C., the mRNA may bepurified using Ambion's MEGACLEAR™ Kit (Austin, Tex.) following themanufacturer's instructions. This kit can purify up to 500 μg of RNA.Following the cleanup, the RNA polynucleotide may be quantified usingthe NANODROP™ and analyzed by agarose gel electrophoresis to confirm theRNA polynucleotide is the proper size and that no degradation of the RNAhas occurred.

Example 5: Enzymatic Capping

Capping of a RNA polynucleotide is performed as follows where themixture includes: IVT RNA 60 μg-180 g and dH₂0 up to 72 μl. The mixtureis incubated at 65° C. for 5 minutes to denature RNA, and then istransferred immediately to ice.

The protocol then involves the mixing of 10× Capping Buffer (0.5 MTris-HCl (pH 8.0), 60 mM KCl, 12.5 mM MgCl₂) (10.0 μl); 20 mM GTP (5.0μl); 20 mM S-Adenosyl Methionine (2.5 μl); RNase Inhibitor (100 U);2′-O-Methyltransferase (400U); Vaccinia capping enzyme (Guanylyltransferase) (40 U); dH₂0 (Up to 28 μl); and incubation at 37° C. for 30minutes for 60 μg RNA or up to 2 hours for 180 μg of RNA.

The RNA polynucleotide may then be purified using Ambion's MEGACLEAR™Kit (Austin, Tex.) following the manufacturer's instructions. Followingthe cleanup, the RNA may be quantified using the NANODROP™(ThermoFisher, Waltham, Mass.) and analyzed by agarose gelelectrophoresis to confirm the RNA polynucleotide is the proper size andthat no degradation of the RNA has occurred. The RNA polynucleotideproduct may also be sequenced by running a reverse-transcription-PCR togenerate the cDNA for sequencing.

Example 6: PolyA Tailing Reaction

Without a poly-T in the cDNA, a poly-A tailing reaction must beperformed before cleaning the final product. This is done by mixingcapped IVT RNA (100 μl); RNase Inhibitor (20 U); 10× Tailing Buffer (0.5M Tris-HCl (pH 8.0), 2.5 M NaCl, 100 mM MgCl₂)(12.0 μl); 20 mM ATP (6.0μl); Poly-A Polymerase (20 U); dH₂0 up to 123.5 μl and incubation at 37°C. for 30 min. If the poly-A tail is already in the transcript, then thetailing reaction may be skipped and proceed directly to cleanup withAmbion's MEGACLEAR™ kit (Austin, Tex.) (up to 500 μg). Poly-A Polymerasemay be a recombinant enzyme expressed in yeast.

It should be understood that the processivity or integrity of the polyAtailing reaction may not always result in an exact size polyA tail.Hence, polyA tails of approximately between 40-200 nucleotides, e.g.,about 40, 50, 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100,101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 150-165, 155, 156,157, 158, 159, 160, 161, 162, 163, 164 or 165 are within the scope ofthe present disclosure.

Example 7: Capping Assays Protein Expression Assay

Polynucleotides (e.g., mRNA) encoding a polypeptide, containing any ofthe caps taught herein, can be transfected into cells at equalconcentrations. The amount of protein secreted into the culture mediumcan be assayed by ELISA at 6, 12, 24 and/or 36 hours post-transfection.Synthetic polynucleotides that secrete higher levels of protein into themedium correspond to a synthetic polynucleotide with a highertranslationally-competent cap structure.

Purity Analysis Synthesis

RNA (e.g., mRNA) polynucleotides encoding a polypeptide, containing anyof the caps taught herein can be compared for purity using denaturingAgarose-Urea gel electrophoresis or HPLC analysis. RNA polynucleotideswith a single, consolidated band by electrophoresis correspond to thehigher purity product compared to polynucleotides with multiple bands orstreaking bands. Chemically modified RNA polynucleotides with a singleHPLC peak also correspond to a higher purity product. The cappingreaction with a higher efficiency provides a more pure polynucleotidepopulation.

Cytokine Analysis

RNA (e.g., mRNA) polynucleotides encoding a polypeptide, containing anyof the caps taught herein can be transfected into cells at multipleconcentrations. The amount of pro-inflammatory cytokines, such asTNF-alpha and IFN-beta, secreted into the culture medium can be assayedby ELISA at 6, 12, 24 and/or 36 hours post-transfection. RNApolynucleotides resulting in the secretion of higher levels ofpro-inflammatory cytokines into the medium correspond to apolynucleotides containing an immune-activating cap structure.

Capping Reaction Efficiency

RNA (e.g., mRNA) polynucleotides encoding a polypeptide, containing anyof the caps taught herein can be analyzed for capping reactionefficiency by LC-MS after nuclease treatment. Nuclease treatment ofcapped polynucleotides yield a mixture of free nucleotides and thecapped 5′-5-triphosphate cap structure detectable by LC-MS. The amountof capped product on the LC-MS spectra can be expressed as a percent oftotal polynucleotide from the reaction and correspond to cappingreaction efficiency. The cap structure with a higher capping reactionefficiency has a higher amount of capped product by LC-MS.

Example 8: Agarose Gel Electrophoresis of Modified RNA or RT PCRProducts

Individual RNA polynucleotides (200-400 ng in a 20 μl volume) or reversetranscribed PCR products (200-400 ng) may be loaded into a well on anon-denaturing 1.2% Agarose E-Gel (Invitrogen, Carlsbad, Calif.) and runfor 12-15 minutes, according to the manufacturer protocol.

Example 9: NANODROP™ Modified RNA Quantification and UV Spectral Data

Chemically modified RNA polynucleotides in TE buffer (1 μl) are used forNANODROP™ UV absorbance readings to quantitate the yield of eachpolynucleotide from an chemical synthesis or in vitro transcriptionreaction.

Example 10: Formulation of Modified mRNA Using Lipidoids

RNA (e.g., mRNA) polynucleotides may be formulated for in vitroexperiments by mixing the polynucleotides with the lipidoid at a setratio prior to addition to cells. In vivo formulation may require theaddition of extra ingredients to facilitate circulation throughout thebody. To test the ability of these lipidoids to form particles suitablefor in vivo work, a standard formulation process used for siRNA-lipidoidformulations may be used as a starting point. After formation of theparticle, polynucleotide is added and allowed to integrate with thecomplex. The encapsulation efficiency is determined using a standard dyeexclusion assays.

Example 11: Exemplary Nucleic Acid Encoding gE RNA Polynucleotide forUse in a VZV Vaccine

The following sequence is an exemplary sequence that can be used toencode a VZV RNA polynucleotide gE for use in a VZV vaccine. A VZVvaccine may comprise, for example, at least one RNA polynucleotideencoded by at least one of the following sequence or by at least onefragment of the following sequence. In some embodiments, the mRNAfurther comprises a 5′ cap, for example, any of the caps disclosedherein, e.g., a cap having sequence m7G(5′)ppp(5′)G-2′-O-methyl. In someembodiments, the mRNA does not have a cap sequence. In some embodiments,the mRNA has at least one chemical modification, for example, any of thechemical modifications disclosed herein, e.g., N1-methylpseudouridinemodification or N1-ethylpseudouridine modification. In otherembodiments, the mRNA does not have chemical modification.

Each of the sequences described herein encompasses a chemically modifiedsequence or an unmodified sequence which includes no modifiednucleotides.

VZV gE-full-length Oka strain: (SEQ ID NO: 1)TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGGGACAGTGAATAAGCCGGTTGTGGGCGTGCTTATGGGCTTTGGGATTATTACCGGTACATTACGAATTACCAATCCAGTGCGCGCCAGTGTGCTGCGTTACGACGACTTTCACATTGACGAGGATAAGCTGGATACTAACAGCGTGTACGAACCTTATTACCACTCAGATCATGCCGAATCAAGCTGGGTTAATAGAGGAGAAAGCAGCCGAAAAGCCTACGACCACAACTCACCTTATATTTGGCCCAGAAACGATTATGACGGTTTCCTGGAAAACGCACATGAACACCATGGAGTCTACAACCAAGGCAGGGGAATCGACAGTGGCGAGCGTCTTATGCAGCCAACACAGATGTCGGCACAGGAGGATCTCGGTGATGACACCGGCATACACGTGATTCCCACATTAAACGGCGACGACAGACATAAGATCGTCAATGTGGATCAGCGTCAGTATGGGGATGTCTTTAAAGGCGATTTGAATCCAAAGCCCCAAGGACAGAGACTGATCGAGGTCTCTGTAGAAGAAAATCACCCCTTCACTTTGCGCGCTCCAATCCAGAGGATTTACGGGGTGCGTTATACCGAAACTTGGAGTTTCTTGCCGTCACTGACGTGTACGGGGGATGCCGCCCCCGCAATCCAGCACATCTGTCTGAAACACACCACATGCTTTCAGGACGTGGTTGTGGATGTGGATTGCGCGGAAAACACAAAAGAAGACCAACTCGCCGAAATCAGCTATCGTTTTCAGGGTAAAAAAGAGGCCGACCAACCGTGGATTGTTGTGAATACGAGCACGCTCTTCGATGAGCTTGAACTCGATCCCCCGGAAATCGAGCCTGGGGTTCTAAAAGTGTTGAGGACCGAGAAGCAGTACCTCGGGGTTTATATCTGGAATATGAGAGGCTCCGATGGCACCTCTACCTACGCAACGTTTCTGGTTACCTGGAAGGGAGACGAGAAGACACGGAATCCAACGCCCGCTGTGACCCCTCAGCCTAGGGGAGCCGAATTCCACATGTGGAACTATCACTCCCATGTATTCAGTGTGGGTGACACTTTCAGCCTGGCCATGCACCTGCAGTATAAGATTCACGAGGCACCCTTCGACCTCCTGCTGGAGTGGTTGTACGTACCTATTGATCCCACTTGTCAGCCCATGCGCCTGTACTCCACTTGCTTGTACCACCCCAATGCACCACAGTGTCTATCACACATGAACTCCGGGTGTACCTTTACTTCACCCCATCTTGCCCAGCGGGTCGCCAGCACAGTGTATCAGAACTGTGAGCATGCTGACAACTATACTGCTTATTGCCTCGGAATATCCCATATGGAGCCAAGCTTCGGGCTCATACTGCACGATGGTGGTACGACACTCAAGTTCGTGGACACCCCCGAAAGCCTTTCTGGCTTGTACGTGTTCGTGGTCTACTTCAATGGACATGTGGAGGCAGTGGCTTACACAGTGGTTTCGACAGTTGATCACTTTGTAAATGCCATTGAGGAACGCGGCTTCCCGCCTACAGCGGGCCAGCCCCCTGCGACAACAAAACCAAAAGAGATTACGCCCGTTAATCCTGGGACTAGTCCATTGCTGAGGTATGCCGCCTGGACTGGCGGTCTGGCGGCCGTGGTACTTCTGTGTTTAGTCATATTTCTGATCTGTACCGCTAAACGTATGCGGGTCAAGGCTTACCGTGTTGACAAGTCTCCTTACAATCAGTCAATGTACTATGCAGGACTCCCTGTTGACGATTTCGAAGACTCAGAGAGTACAGACACAGAAGAAGAATTCGGAAACGCTATAGGTGGCTCTCACGGAGGTAGCTCGTATACAGTGTACATCGATAAAACCAGATGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCVZV gE-full-length Oka strain (mRNA): (SEQ ID NO: 123)UCAAGCUUUUGGACCCUCGUACAGAAGCUAAUACGACUCACUAUAGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGGGGACAGUGAAUAAGCCGGUUGUGGGCGUGCUUAUGGGCUUUGGGAUUAUUACCGGUACAUUACGAAUUACCAAUCCAGUGCGCGCCAGUGUGCUGCGUUACGACGACUUUCACAUUGACGAGGAUAAGCUGGAUACUAACAGCGUGUACGAACCUUAUUACCACUCAGAUCAUGCCGAAUCAAGCUGGGUUAAUAGAGGAGAAAGCAGCCGAAAAGCCUACGACCACAACUCACCUUAUAUUUGGCCCAGAAACGAUUAUGACGGUUUCCUGGAAAACGCACAUGAACACCAUGGAGUCUACAACCAAGGCAGGGGAAUCGACAGUGGCGAGCGUCUUAUGCAGCCAACACAGAUGUCGGCACAGGAGGAUCUCGGUGAUGACACCGGCAUACACGUGAUUCCCACAUUAAACGGCGACGACAGACAUAAGAUCGUCAAUGUGGAUCAGCGUCAGUAUGGGGAUGUCUUUAAAGGCGAUUUGAAUCCAAAGCCCCAAGGACAGAGACUGAUCGAGGUCUCUGUAGAAGAAAAUCACCCCUUCACUUUGCGCGCUCCAAUCCAGAGGAUUUACGGGGUGCGUUAUACCGAAACUUGGAGUUUCUUGCCGUCACUGACGUGUACGGGGGAUGCCGCCCCCGCAAUCCAGCACAUCUGUCUGAAACACACCACAUGCUUUCAGGACGUGGUUGUGGAUGUGGAUUGCGCGGAAAACACAAAAGAAGACCAACUCGCCGAAAUCAGCUAUCGUUUUCAGGGUAAAAAAGAGGCCGACCAACCGUGGAUUGUUGUGAAUACGAGCACGCUCUUCGAUGAGCUUGAACUCGAUCCCCCGGAAAUCGAGCCUGGGGUUCUAAAAGUGUUGAGGACCGAGAAGCAGUACCUCGGGGUUUAUAUCUGGAAUAUGAGAGGCUCCGAUGGCACCUCUACCUACGCAACGUUUCUGGUUACCUGGAAGGGAGACGAGAAGACACGGAAUCCAACGCCCGCUGUGACCCCUCAGCCUAGGGGAGCCGAAUUCCACAUGUGGAACUAUCACUCCCAUGUAUUCAGUGUGGGUGACACUUUCAGCCUGGCCAUGCACCUGCAGUAUAAGAUUCACGAGGCACCCUUCGACCUCCUGCUGGAGUGGUUGUACGUACCUAUUGAUCCCACUUGUCAGCCCAUGCGCCUGUACUCCACUUGCUUGUACCACCCCAAUGCACCACAGUGUCUAUCACACAUGAACUCCGGGUGUACCUUUACUUCACCCCAUCUUGCCCAGCGGGUCGCCAGCACAGUGUAUCAGAACUGUGAGCAUGCUGACAACUAUACUGCUUAUUGCCUCGGAAUAUCCCAUAUGGAGCCAAGCUUCGGGCUCAUACUGCACGAUGGUGGUACGACACUCAAGUUCGUGGACACCCCCGAAAGCCUUUCUGGCUUGUACGUGUUCGUGGUCUACUUCAAUGGACAUGUGGAGGCAGUGGCUUACACAGUGGUUUCGACAGUUGAUCACUUUGUAAAUGCCAUUGAGGAACGCGGCUUCCCGCCUACAGCGGGCCAGCCCCCUGCGACAACAAAACCAAAAGAGAUUACGCCCGUUAAUCCUGGGACUAGUCCAUUGCUGAGGUAUGCCGCCUGGACUGGCGGUCUGGCGGCCGUGGUACUUCUGUGUUUAGUCAUAUUUCUGAUCUGUACCGCUAAACGUAUGCGGGUCAAGGCUUACCGUGUUGACAAGUCUCCUUACAAUCAGUCAAUGUACUAUGCAGGACUCCCUGUUGACGAUUUCGAAGACUCAGAGAGUACAGACACAGAAGAAGAAUUCGGAAACGCUAUAGGUGGCUCUCACGGAGGUAGCUCGUAUACAGUGUACAUCGAUAAAACCAGAUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC

Example 12: Exemplary Nucleic Acid Encoding gI RNA Polynucleotide forUse in a VZV Vaccine

The following sequence is an exemplary sequence that can be used toencode a VZV RNA polynucleotide gI for use in a VZV RNA (e.g., mRNA)vaccine. The gI polypeptide forms a complex with gE in infected cellswhich facilitates the endocytosis of both glycoproteins and directs themto the trans-Golgi network (TGN) where the final viral envelope isacquired. A VZV vaccine may comprise, for example, at least one RNA(e.g., mRNA) polynucleotide encoded by at least one of the followingsequence or by at least one fragment of the following sequence. In someembodiments, the mRNA further comprises a 5′ cap, for example, any ofthe caps disclosed herein, e.g., a cap having sequencem7G(5′)ppp(5′)G-2′-O-methyl. In other embodiments, the mRNA does nothave a cap sequence. In some embodiments, the mRNA has at least onechemical modification, for example, any of the chemical modificationsdisclosed herein, e.g., N1-methylpseudouridine modification orN1-ethylpseudouridine modification. In other embodiments, the mRNA doesnot have chemical modification.

VZV-GI-full length (Oka strain): (SEQ ID NO: 2)TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGTTTTTAATCCAATGTTTGATATCGGCCGTTATATTTTACATACAAGTGACCAACGCTTTGATCTTCAAGGGCGACCACGTGAGCTTGCAAGTTAACAGCAGTCTCACGTCTATCCTTATTCCCATGCAAAATGATAATTATACAGAGATAAAAGGACAGCTTGTCTTTATTGGAGAGCAACTACCTACCGGGACAAACTATAGCGGAACACTGGAACTGTTATACGCGGATACGGTGGCGTTTTGTTTCCGGTCAGTACAAGTAATAAGATACGACGGATGTCCCCGGATTAGAACGAGCGCTTTTATTTCGTGTAGGTACAAACATTCGTGGCATTATGGTAACTCAACGGATCGGATATCAACAGAGCCGGATGCTGGTGTAATGTTGAAAATTACCAAACCGGGAATAAATGATGCTGGTGTGTATGTACTTCTTGTTCGGTTAGACCATAGCAGATCCACCGATGGTTTCATTCTTGGTGTAAATGTATATACAGCGGGCTCGCATCACAACATTCACGGGGTTATCTACACTTCTCCATCTCTACAGAATGGATATTCTACAAGAGCCCTTTTTCAACAAGCTCGTTTGTGTGATTTACCCGCGACACCCAAAGGGTCCGGTACCTCCCTGTTTCAACATATGCTTGATCTTCGTGCCGGTAAATCGTTAGAGGATAACCCTTGGTTACATGAGGACGTTGTTACGACAGAAACTAAGTCCGTTGTTAAGGAGGGGATAGAAAATCACGTATATCCAACGGATATGTCCACGTTACCCGAAAAGTCCCTTAATGATCCTCCAGAAAATCTACTTATAATTATTCCTATAGTAGCGTCTGTCATGATCCTCACCGCCATGGTTATTGTTATTGTAATAAGCGTTAAGCGACGTAGAATTAAAAAACATCCAATTTATCGCCCAAATACAAAAACAAGAAGGGGCATACAAAATGCGACACCAGAATCCGATGTGATGTTGGAGGCCGCCATTGCACAACTAGCAACGATTCGCGAAGAATCCCCCCCACATTCCGTTGTAAACCCGTTTGTTAAATAGTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC VZV-GI-full length (Oka strain)(mRNA):(SEQ ID NO: 124)UCAAGCUUUUGGACCCUCGUACAGAAGCUAAUACGACUCACUAUAGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGUUUUUAAUCCAAUGUUUGAUAUCGGCCGUUAUAUUUUACAUACAAGUGACCAACGCUUUGAUCUUCAAGGGCGACCACGUGAGCUUGCAAGUUAACAGCAGUCUCACGUCUAUCCUUAUUCCCAUGCAAAAUGAUAAUUAUACAGAGAUAAAAGGACAGCUUGUCUUUAUUGGAGAGCAACUACCUACCGGGACAAACUAUAGCGGAACACUGGAACUGUUAUACGCGGAUACGGUGGCGUUUUGUUUCCGGUCAGUACAAGUAAUAAGAUACGACGGAUGUCCCCGGAUUAGAACGAGCGCUUUUAUUUCGUGUAGGUACAAACAUUCGUGGCAUUAUGGUAACUCAACGGAUCGGAUAUCAACAGAGCCGGAUGCUGGUGUAAUGUUGAAAAUUACCAAACCGGGAAUAAAUGAUGCUGGUGUGUAUGUACUUCUUGUUCGGUUAGACCAUAGCAGAUCCACCGAUGGUUUCAUUCUUGGUGUAAAUGUAUAUACAGCGGGCUCGCAUCACAACAUUCACGGGGUUAUCUACACUUCUCCAUCUCUACAGAAUGGAUAUUCUACAAGAGCCCUUUUUCAACAAGCUCGUUUGUGUGAUUUACCCGCGACACCCAAAGGGUCCGGUACCUCCCUGUUUCAACAUAUGCUUGAUCUUCGUGCCGGUAAAUCGUUAGAGGAUAACCCUUGGUUACAUGAGGACGUUGUUACGACAGAAACUAAGUCCGUUGUUAAGGAGGGGAUAGAAAAUCACGUAUAUCCAACGGAUAUGUCCACGUUACCCGAAAAGUCCCUUAAUGAUCCUCCAGAAAAUCUACUUAUAAUUAUUCCUAUAGUAGCGUCUGUCAUGAUCCUCACCGCCAUGGUUAUUGUUAUUGUAAUAAGCGUUAAGCGACGUAGAAUUAAAAAACAUCCAAUUUAUCGCCCAAAUACAAAAACAAGAAGGGGCAUACAAAAUGCGACACCAGAAUCCGAUGUGAUGUUGGAGGCCGCCAUUGCACAACUAGCAACGAUUCGCGAAGAAUCCCCCCCACAUUCCGUUGUAAACCCGUUUGUUAAAUAGUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC

Example 13: mRNAs Encoding Variant gE Antigens Having DifferentC-Terminal Sequence for Use in a VZV Vaccine

VZV is enveloped in the trans-golgi network. Glycoprotein I(gI) forms acomplex with gE in infected cells which facilitates the endocytosis ofboth glycoproteins and directs them to the trans-Golgi network (TGN)where the final viral envelope is acquired. mRNAs encoding gE antigenshaving different C-terminal variant sequence were designed to avoid gEbeing trapped in the ER/golgi/TGN, leading to an increase in thelocalization of gE antigen to the plasma membrane and improvedimmune-stimulating capabilities. A schematic of the gE antigen is shownin FIG. 4.

Several different gE variant mRNA sequences (Oka strain) wereengineered:

(1) gE variant mRNA encoding a truncated polypeptide having the terminal62 amino acids of the C terminal region deleted (SEQ ID NO: 17-20). Theresultant polypeptide has reduced localization to the trans-golginetwork and reduced endocytosis.(2) gE variant mRNA encoding a truncated polypeptide having the terminal62 amino acids of the C terminal region deleted and also having thesignal peptide replaced with IgKappa, which results in a secreted formof the truncated gE polypeptide (SEQ ID NO: 21-24). The resultantpolypeptide has reduced localization to the trans-golgi network andreduced endocytosis.(3) gE variant mRNA encoding a truncated polypeptide having the terminal50 amino acids of the C terminal region deleted (SEQ ID NO: 33-36). Theresultant polypeptide has reduced localization to the trans-golginetwork and reduced endocytosis.(4) gE variant mRNA encoding a truncated polypeptide having the terminal50 amino acids of the C terminal region deleted and also having thepoint mutation Y569A (SEQ ID NO: 37-40). The “AYRV” motif (SEQ ID NO:119) is a trafficking motif which targets the gE polypeptide to thetrans-golgi network. Thus, mutating the AARV sequence SEQ ID NO: 120 toAYRV SEQ ID NO: 119 results in reduced localization of the gEpolypeptide to the trans-golgi network.(5) gE variant mRNA encoding full-length gE polypeptide with an AEAADA(SEQ ID NO: 58) sequence (SEQ ID NO: 25-28). The A-E-A-A-D-A (SEQ ID NO:58) sequence replaces SESTDT (SEQ ID NO: 59). This is a replacement ofthe Ser/Thr-rich “SSTT” (SEQ ID NO: 122) acidic cluster with an Ala-richsequence. This reduces CKII phosphorylation, which in turn results inreduced localization of the gE polypeptide to the trans-golgi network.(6) gE variant mRNA encoding full-length gE polypeptide with an AEAADA(SEQ ID NO: 58) sequence and also having the point mutation Y582G (SEQID NO: 29-32). The “YAGL” (SEQ ID NO: 121) motif is an endocytosis motifwhich enhances localization of the gE polypeptide to the trans-golginetwork. Thus, mutating the GAGL sequence (SEQ ID NO: 132) to YAGL (SEQID NO: 121) results in reduced endocytosis of the resultant polypeptide.

Each of these variants have modifications that reduce localization ofthe encoded gE protein to the trans-golgi network and enhancetrafficking to the plasma membrane. Table 1 summarizes mRNAs encodingthe variant gE antigens having different C-terminal sequence. In someembodiments, the variant mRNA further comprises a 5′ cap, for example,any of the caps disclosed herein, e.g., a cap having sequencem7G(5′)ppp(5′)G-2′-O-methyl. In some embodiments, the variant mRNA doesnot have a 5′ cap. In some embodiments, the variant mRNA has at leastone chemical modification, for example, any of the chemicalmodifications disclosed herein, e.g., N1-methylpseudouridinemodification or N1-ethylpseudouridine modification. In some embodiments,the mRNA does not have chemical modification. The sequences encoding themRNA variants are provided beneath the table.

TABLE 1 mRNA Constructs Name of mRNA SEQ ID NO: construct DescriptionFunction 3 (DNA) and VZV-GE- Truncated VZV gE The C-terminal 125 (mRNA)delete-562 sequence-deletion sequence targets from gE to the trans- aa562 (62 aa Golgi network deletion from C (TGN); truncation terminaldomain) assists in reducing gE localization to TGN 4 (DNA) and VZV-GE-Secreted form of The C-terminal 126 (mRNA) delete-562- truncatedsequence targets IgKappa VZV gE sequence- gE to the trans- deletion fromaa 562 Golgi networks (62 aa deletion from (TGN); truncation C terminaldomain) assists in and signal reducing gE peptide replaced localizationto with IgKappa TGN 5 (DNA) and VZV-GE- Truncated VZV gE The C-terminal127 (mRNA) delete-574 sequence-deletion sequence targets from gE to theaa 574 (50 aa trans-Golgi deletion from C network (TGN); terminaldomain) truncation assists in reducing gE localization to TGN 6 (DNA)and VZV-GE- Truncated VZV gE The C-terminal 128 (mRNA) delete-574-sequence-deletion sequence targets Y569A from gE to the trans- aa 574(50 aa Golgi network deletion from C (TGN); the AYRV terminal domain)(SEQ ID NO: and Y569 A 119) sequence point mutation is required fortargeting gE to the TGN; truncation/ mutation reduces localization toTGN) 7 (DNA) and VZV-GE-full- VZV gE full length AEAADA (SEQ 129 (mRNA)length- sequence ID NO: 58) AEAADA with AEAADA replaces SSTT (SEQ ID NO:58) (SEQ ID NO: 58) (SEQ ID NO: 122) sequence (acid cluster) comprisinga phosphorylation motif, which phosphorylation assists in localizing gEto the TGN; mutation reduces localization of gE to TGN 8 (DNA) andVZV-GE-full- VZV gE-full length Mutations assist 130 (mRNA) AEAADAlength- sequence with in reducing (SEQ ID NO: 58)- AEAADA sequenceendocytosis and Y582G (SEQ ID NO: 58) localization and Y582G of gE pointmutation to the TGN

VZV-GE-delete-562 (SEQ ID NO: 3)TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGGGACAGTTAATAAACCTGTGGTGGGGGTATTGATGGGGTTCGGAATTATCACGGGAACGTTGCGTATAACGAATCCGGTCAGAGCATCCGTCTTGCGATACGATGATTTTCACATCGATGAAGACAAACTGGATACAAACTCCGTATATGAGCCTTACTACCATTCAGATCATGCGGAGTCTTCATGGGTAAATCGGGGAGAGTCTTCGCGAAAAGCGTACGATCATAACTCACCTTATATATGGCCACGTAATGATTATGATGGATTTTTAGAGAACGCACACGAACACCATGGGGTGTATAATCAGGGCCGTGGTATCGATAGCGGGGAACGGTTAATGCAACCCACACAAATGTCTGCACAGGAGGATCTTGGGGACGATACGGGCATCCACGTTATCCCTACGTTAAACGGCGATGACAGACATAAAATTGTAAATGTGGACCAACGTCAATACGGTGACGTGTTTAAAGGAGATCTTAATCCAAAACCCCAAGGCCAAAGACTCATTGAGGTGTCAGTGGAAGAAAATCACCCGTTTACTTTACGCGCACCGATTCAGCGGATTTATGGAGTCCGGTACACCGAGACTTGGAGCTTTTTGCCGTCATTAACCTGTACGGGAGACGCAGCGCCCGCCATCCAGCATATATGTTTAAAACATACAACATGCTTTCAAGACGTGGTGGTGGATGTGGATTGCGCGGAAAATACTAAAGAGGATCAGTTGGCCGAAATCAGTTACCGTTTTCAAGGTAAGAAGGAAGCGGACCAACCGTGGATTGTTGTAAACACGAGCACACTGTTTGATGAACTCGAATTAGACCCCCCCGAGATTGAACCGGGTGTCTTGAAAGTACTTCGGACAGAAAAACAATACTTGGGTGTGTACATTTGGAACATGCGCGGCTCCGATGGTACGTCTACCTACGCCACGTTTTTGGTCACCTGGAAAGGGGATGAAAAAACAAGAAACCCTACGCCCGCAGTAACTCCTCAACCAAGAGGGGCTGAGTTTCATATGTGGAATTACCACTCGCATGTATTTTCAGTTGGTGATACGTTTAGCTTGGCAATGCATCTTCAGTATAAGATACATGAAGCGCCATTTGATTTGCTGTTAGAGTGGTTGTATGTCCCCATCGATCCTACATGTCAACCAATGCGGTTATATTCTACGTGTTTGTATCATCCCAACGCACCCCAATGCCTCTCTCATATGAATTCCGGTTGTACATTTACCTCGCCACATTTAGCCCAGCGTGTTGCAAGCACAGTGTATCAAAATTGTGAACATGCAGATAACTACACCGCATATTGTCTGGGAATATCTCATATGGAGCCTAGCTTTGGTCTAATCTTACACGACGGGGGCACCACGTTAAAGTTTGTAGATACACCCGAGAGTTTGTCGGGATTATACGTTTTTGTGGTGTATTTTAACGGGCATGTTGAAGCCGTAGCATACACTGTTGTATCCACAGTAGATCATTTTGTAAACGCAATTGAAGAGCGTGGATTTCCGCCAACGGCCGGTCAGCCACCGGCGACTACTAAACCCAAGGAAATTACCCCCGTAAACCCCGGAACGTCACCACTTCTACGATATGCCGCATGGACCGGAGGGCTTGCAGCAGTAGTACTTTTATGTCTCGTAATATTTTTAATCTGTACGGCTTGATGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCVZV-GE-delete-562 (mRNA) (SEQ ID NO: 125)UCAAGCUUUUGGACCCUCGUACAGAAGCUAAUACGACUCACUAUAGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGGGGACAGUUAAUAAACCUGUGGUGGGGGUAUUGAUGGGGUUCGGAAUUAUCACGGGAACGUUGCGUAUAACGAAUCCGGUCAGAGCAUCCGUCUUGCGAUACGAUGAUUUUCACAUCGAUGAAGACAAACUGGAUACAAACUCCGUAUAUGAGCCUUACUACCAUUCAGAUCAUGCGGAGUCUUCAUGGGUAAAUCGGGGAGAGUCUUCGCGAAAAGCGUACGAUCAUAACUCACCUUAUAUAUGGCCACGUAAUGAUUAUGAUGGAUUUUUAGAGAACGCACACGAACACCAUGGGGUGUAUAAUCAGGGCCGUGGUAUCGAUAGCGGGGAACGGUUAAUGCAACCCACACAAAUGUCUGCACAGGAGGAUCUUGGGGACGAUACGGGCAUCCACGUUAUCCCUACGUUAAACGGCGAUGACAGACAUAAAAUUGUAAAUGUGGACCAACGUCAAUACGGUGACGUGUUUAAAGGAGAUCUUAAUCCAAAACCCCAAGGCCAAAGACUCAUUGAGGUGUCAGUGGAAGAAAAUCACCCGUUUACUUUACGCGCACCGAUUCAGCGGAUUUAUGGAGUCCGGUACACCGAGACUUGGAGCUUUUUGCCGUCAUUAACCUGUACGGGAGACGCAGCGCCCGCCAUCCAGCAUAUAUGUUUAAAACAUACAACAUGCUUUCAAGACGUGGUGGUGGAUGUGGAUUGCGCGGAAAAUACUAAAGAGGAUCAGUUGGCCGAAAUCAGUUACCGUUUUCAAGGUAAGAAGGAAGCGGACCAACCGUGGAUUGUUGUAAACACGAGCACACUGUUUGAUGAACUCGAAUUAGACCCCCCCGAGAUUGAACCGGGUGUCUUGAAAGUACUUCGGACAGAAAAACAAUACUUGGGUGUGUACAUUUGGAACAUGCGCGGCUCCGAUGGUACGUCUACCUACGCCACGUUUUUGGUCACCUGGAAAGGGGAUGAAAAAACAAGAAACCCUACGCCCGCAGUAACUCCUCAACCAAGAGGGGCUGAGUUUCAUAUGUGGAAUUACCACUCGCAUGUAUUUUCAGUUGGUGAUACGUUUAGCUUGGCAAUGCAUCUUCAGUAUAAGAUACAUGAAGCGCCAUUUGAUUUGCUGUUAGAGUGGUUGUAUGUCCCCAUCGAUCCUACAUGUCAACCAAUGCGGUUAUAUUCUACGUGUUUGUAUCAUCCCAACGCACCCCAAUGCCUCUCUCAUAUGAAUUCCGGUUGUACAUUUACCUCGCCACAUUUAGCCCAGCGUGUUGCAAGCACAGUGUAUCAAAAUUGUGAACAUGCAGAUAACUACACCGCAUAUUGUCUGGGAAUAUCUCAUAUGGAGCCUAGCUUUGGUCUAAUCUUACACGACGGGGGCACCACGUUAAAGUUUGUAGAUACACCCGAGAGUUUGUCGGGAUUAUACGUUUUUGUGGUGUAUUUUAACGGGCAUGUUGAAGCCGUAGCAUACACUGUUGUAUCCACAGUAGAUCAUUUUGUAAACGCAAUUGAAGAGCGUGGAUUUCCGCCAACGGCCGGUCAGCCACCGGCGACUACUAAACCCAAGGAAAUUACCCCCGUAAACCCCGGAACGUCACCACUUCUACGAUAUGCCGCAUGGACCGGAGGGCUUGCAGCAGUAGUACUUUUAUGUCUCGUAAUAUUUUUAAUCUGUACGGCUUGAUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCG GCVZV-GE-delete-562-IgKappa (SEQ ID NO: 4)TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGAAACCCCGGCGCAGCTGCTGTTTCTGCTGCTGCTGTGGCTGCCGGATACCACCGGCTCCGTCTTGCGATACGATGATTTTCACATCGATGAAGACAAACTGGATACAAACTCCGTATATGAGCCTTACTACCATTCAGATCATGCGGAGTCTTCATGGGTAAATCGGGGAGAGTCTTCGCGAAAAGCGTACGATCATAACTCACCTTATATATGGCCACGTAATGATTATGATGGATTTTTAGAGAACGCACACGAACACCATGGGGTGTATAATCAGGGCCGTGGTATCGATAGCGGGGAACGGTTAATGCAACCCACACAAATGTCTGCACAGGAGGATCTTGGGGACGATACGGGCATCCACGTTATCCCTACGTTAAACGGCGATGACAGACATAAAATTGTAAATGTGGACCAACGTCAATACGGTGACGTGTTTAAAGGAGATCTTAATCCAAAACCCCAAGGCCAAAGACTCATTGAGGTGTCAGTGGAAGAAAATCACCCGTTTACTTTACGCGCACCGATTCAGCGGATTTATGGAGTCCGGTACACCGAGACTTGGAGCTTTTTGCCGTCATTAACCTGTACGGGAGACGCAGCGCCCGCCATCCAGCATATATGTTTAAAACATACAACATGCTTTCAAGACGTGGTGGTGGATGTGGATTGCGCGGAAAATACTAAAGAGGATCAGTTGGCCGAAATCAGTTACCGTTTTCAAGGTAAGAAGGAAGCGGACCAACCGTGGATTGTTGTAAACACGAGCACACTGTTTGATGAACTCGAATTAGACCCCCCCGAGATTGAACCGGGTGTCTTGAAAGTACTTCGGACAGAAAAACAATACTTGGGTGTGTACATTTGGAACATGCGCGGCTCCGATGGTACGTCTACCTACGCCACGTTTTTGGTCACCTGGAAAGGGGATGAAAAAACAAGAAACCCTACGCCCGCAGTAACTCCTCAACCAAGAGGGGCTGAGTTTCATATGTGGAATTACCACTCGCATGTATTTTCAGTTGGTGATACGTTTAGCTTGGCAATGCATCTTCAGTATAAGATACATGAAGCGCCATTTGATTTGCTGTTAGAGTGGTTGTATGTCCCCATCGATCCTACATGTCAACCAATGCGGTTATATTCTACGTGTTTGTATCATCCCAACGCACCCCAATGCCTCTCTCATATGAATTCCGGTTGTACATTTACCTCGCCACATTTAGCCCAGCGTGTTGCAAGCACAGTGTATCAAAATTGTGAACATGCAGATAACTACACCGCATATTGTCTGGGAATATCTCATATGGAGCCTAGCTTTGGTCTAATCTTACACGACGGGGGCACCACGTTAAAGTTTGTAGATACACCCGAGAGTTTGTCGGGATTATACGTTTTTGTGGTGTATTTTAACGGGCATGTTGAAGCCGTAGCATACACTGTTGTATCCACAGTAGATCATTTTGTAAACGCAATTGAAGAGCGTGGATTTCCGCCAACGGCCGGTCAGCCACCGGCGACTACTAAACCCAAGGAAATTACCCCCGTAAACCCCGGAACGTCACCACTTCTACGATATGCCGCATGGACCGGAGGGCTTGCAGCAGTAGTACTTTTATGTCTCGTAATATTTTTAATCTGTACGGCTTGATGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC VZV-GE-delete-562-IgKappa (mRNA)(SEQ ID NO: 126)UCAAGCUUUUGGACCCUCGUACAGAAGCUAAUACGACUCACUAUAGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGGAAACCCCGGCGCAGCUGCUGUUUCUGCUGCUGCUGUGGCUGCCGGAUACCACCGGCUCCGUCUUGCGAUACGAUGAUUUUCACAUCGAUGAAGACAAACUGGAUACAAACUCCGUAUAUGAGCCUUACUACCAUUCAGAUCAUGCGGAGUCUUCAUGGGUAAAUCGGGGAGAGUCUUCGCGAAAAGCGUACGAUCAUAACUCACCUUAUAUAUGGCCACGUAAUGAUUAUGAUGGAUUUUUAGAGAACGCACACGAACACCAUGGGGUGUAUAAUCAGGGCCGUGGUAUCGAUAGCGGGGAACGGUUAAUGCAACCCACACAAAUGUCUGCACAGGAGGAUCUUGGGGACGAUACGGGCAUCCACGUUAUCCCUACGUUAAACGGCGAUGACAGACAUAAAAUUGUAAAUGUGGACCAACGUCAAUACGGUGACGUGUUUAAAGGAGAUCUUAAUCCAAAACCCCAAGGCCAAAGACUCAUUGAGGUGUCAGUGGAAGAAAAUCACCCGUUUACUUUACGCGCACCGAUUCAGCGGAUUUAUGGAGUCCGGUACACCGAGACUUGGAGCUUUUUGCCGUCAUUAACCUGUACGGGAGACGCAGCGCCCGCCAUCCAGCAUAUAUGUUUAAAACAUACAACAUGCUUUCAAGACGUGGUGGUGGAUGUGGAUUGCGCGGAAAAUACUAAAGAGGAUCAGUUGGCCGAAAUCAGUUACCGUUUUCAAGGUAAGAAGGAAGCGGACCAACCGUGGAUUGUUGUAAACACGAGCACACUGUUUGAUGAACUCGAAUUAGACCCCCCCGAGAUUGAACCGGGUGUCUUGAAAGUACUUCGGACAGAAAAACAAUACUUGGGUGUGUACAUUUGGAACAUGCGCGGCUCCGAUGGUACGUCUACCUACGCCACGUUUUUGGUCACCUGGAAAGGGGAUGAAAAAACAAGAAACCCUACGCCCGCAGUAACUCCUCAACCAAGAGGGGCUGAGUUUCAUAUGUGGAAUUACCACUCGCAUGUAUUUUCAGUUGGUGAUACGUUUAGCUUGGCAAUGCAUCUUCAGUAUAAGAUACAUGAAGCGCCAUUUGAUUUGCUGUUAGAGUGGUUGUAUGUCCCCAUCGAUCCUACAUGUCAACCAAUGCGGUUAUAUUCUACGUGUUUGUAUCAUCCCAACGCACCCCAAUGCCUCUCUCAUAUGAAUUCCGGUUGUACAUUUACCUCGCCACAUUUAGCCCAGCGUGUUGCAAGCACAGUGUAUCAAAAUUGUGAACAUGCAGAUAACUACACCGCAUAUUGUCUGGGAAUAUCUCAUAUGGAGCCUAGCUUUGGUCUAAUCUUACACGACGGGGGCACCACGUUAAAGUUUGUAGAUACACCCGAGAGUUUGUCGGGAUUAUACGUUUUUGUGGUGUAUUUUAACGGGCAUGUUGAAGCCGUAGCAUACACUGUUGUAUCCACAGUAGAUCAUUUUGUAAACGCAAUUGAAGAGCGUGGAUUUCCGCCAACGGCCGGUCAGCCACCGGCGACUACUAAACCCAAGGAAAUUACCCCCGUAAACCCCGGAACGUCACCACUUCUACGAUAUGCCGCAUGGACCGGAGGGCUUGCAGCAGUAGUACUUUUAUGUCUCGUAAUAUUUUUAAUCUGUACGGCUUGAUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC VZV-GE- delete-574 (SEQ ID NO: 5)TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGGGACAGTTAATAAACCTGTGGTGGGGGTATTGATGGGGTTCGGAATTATCACGGGAACGTTGCGTATAACGAATCCGGTCAGAGCATCCGTCTTGCGATACGATGATTTTCACATCGATGAAGACAAACTGGATACAAACTCCGTATATGAGCCTTACTACCATTCAGATCATGCGGAGTCTTCATGGGTAAATCGGGGAGAGTCTTCGCGAAAAGCGTACGATCATAACTCACCTTATATATGGCCACGTAATGATTATGATGGATTTTTAGAGAACGCACACGAACACCATGGGGTGTATAATCAGGGCCGTGGTATCGATAGCGGGGAACGGTTAATGCAACCCACACAAATGTCTGCACAGGAGGATCTTGGGGACGATACGGGCATCCACGTTATCCCTACGTTAAACGGCGATGACAGACATAAAATTGTAAATGTGGACCAACGTCAATACGGTGACGTGTTTAAAGGAGATCTTAATCCAAAACCCCAAGGCCAAAGACTCATTGAGGTGTCAGTGGAAGAAAATCACCCGTTTACTTTACGCGCACCGATTCAGCGGATTTATGGAGTCCGGTACACCGAGACTTGGAGCTTTTTGCCGTCATTAACCTGTACGGGAGACGCAGCGCCCGCCATCCAGCATATATGTTTAAAACATACAACATGCTTTCAAGACGTGGTGGTGGATGTGGATTGCGCGGAAAATACTAAAGAGGATCAGTTGGCCGAAATCAGTTACCGTTTTCAAGGTAAGAAGGAAGCGGACCAACCGTGGATTGTTGTAAACACGAGCACACTGTTTGATGAACTCGAATTAGACCCCCCCGAGATTGAACCGGGTGTCTTGAAAGTACTTCGGACAGAAAAACAATACTTGGGTGTGTACATTTGGAACATGCGCGGCTCCGATGGTACGTCTACCTACGCCACGTTTTTGGTCACCTGGAAAGGGGATGAAAAAACAAGAAACCCTACGCCCGCAGTAACTCCTCAACCAAGAGGGGCTGAGTTTCATATGTGGAATTACCACTCGCATGTATTTTCAGTTGGTGATACGTTTAGCTTGGCAATGCATCTTCAGTATAAGATACATGAAGCGCCATTTGATTTGCTGTTAGAGTGGTTGTATGTCCCCATCGATCCTACATGTCAACCAATGCGGTTATATTCTACGTGTTTGTATCATCCCAACGCACCCCAATGCCTCTCTCATATGAATTCCGGTTGTACATTTACCTCGCCACATTTAGCCCAGCGTGTTGCAAGCACAGTGTATCAAAATTGTGAACATGCAGATAACTACACCGCATATTGTCTGGGAATATCTCATATGGAGCCTAGCTTTGGTCTAATCTTACACGACGGGGGCACCACGTTAAAGTTTGTAGATACACCCGAGAGTTTGTCGGGATTATACGTTTTTGTGGTGTATTTTAACGGGCATGTTGAAGCCGTAGCATACACTGTTGTATCCACAGTAGATCATTTTGTAAACGCAATTGAAGAGCGTGGATTTCCGCCAACGGCCGGTCAGCCACCGGCGACTACTAAACCCAAGGAAATTACCCCCGTAAACCCCGGAACGTCACCACTTCTACGATATGCCGCATGGACCGGAGGGCTTGCAGCAGTAGTACTTTTATGTCTCGTAATATTTTTAATCTGTACGGCTAAACGAATGAGGGTTAAAGCCTATAGGGTAGACAAGTGATGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC VZV-GE- delete-574 (mRNA) (SEQ ID NO: 127)UCAAGCUUUUGGACCCUCGUACAGAAGCUAAUACGACUCACUAUAGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGGGGACAGUUAAUAAACCUGUGGUGGGGGUAUUGAUGGGGUUCGGAAUUAUCACGGGAACGUUGCGUAUAACGAAUCCGGUCAGAGCAUCCGUCUUGCGAUACGAUGAUUUUCACAUCGAUGAAGACAAACUGGAUACAAACUCCGUAUAUGAGCCUUACUACCAUUCAGAUCAUGCGGAGUCUUCAUGGGUAAAUCGGGGAGAGUCUUCGCGAAAAGCGUACGAUCAUAACUCACCUUAUAUAUGGCCACGUAAUGAUUAUGAUGGAUUUUUAGAGAACGCACACGAACACCAUGGGGUGUAUAAUCAGGGCCGUGGUAUCGAUAGCGGGGAACGGUUAAUGCAACCCACACAAAUGUCUGCACAGGAGGAUCUUGGGGACGAUACGGGCAUCCACGUUAUCCCUACGUUAAACGGCGAUGACAGACAUAAAAUUGUAAAUGUGGACCAACGUCAAUACGGUGACGUGUUUAAAGGAGAUCUUAAUCCAAAACCCCAAGGCCAAAGACUCAUUGAGGUGUCAGUGGAAGAAAAUCACCCGUUUACUUUACGCGCACCGAUUCAGCGGAUUUAUGGAGUCCGGUACACCGAGACUUGGAGCUUUUUGCCGUCAUUAACCUGUACGGGAGACGCAGCGCCCGCCAUCCAGCAUAUAUGUUUAAAACAUACAACAUGCUUUCAAGACGUGGUGGUGGAUGUGGAUUGCGCGGAAAAUACUAAAGAGGAUCAGUUGGCCGAAAUCAGUUACCGUUUUCAAGGUAAGAAGGAAGCGGACCAACCGUGGAUUGUUGUAAACACGAGCACACUGUUUGAUGAACUCGAAUUAGACCCCCCCGAGAUUGAACCGGGUGUCUUGAAAGUACUUCGGACAGAAAAACAAUACUUGGGUGUGUACAUUUGGAACAUGCGCGGCUCCGAUGGUACGUCUACCUACGCCACGUUUUUGGUCACCUGGAAAGGGGAUGAAAAAACAAGAAACCCUACGCCCGCAGUAACUCCUCAACCAAGAGGGGCUGAGUUUCAUAUGUGGAAUUACCACUCGCAUGUAUUUUCAGUUGGUGAUACGUUUAGCUUGGCAAUGCAUCUUCAGUAUAAGAUACAUGAAGCGCCAUUUGAUUUGCUGUUAGAGUGGUUGUAUGUCCCCAUCGAUCCUACAUGUCAACCAAUGCGGUUAUAUUCUACGUGUUUGUAUCAUCCCAACGCACCCCAAUGCCUCUCUCAUAUGAAUUCCGGUUGUACAUUUACCUCGCCACAUUUAGCCCAGCGUGUUGCAAGCACAGUGUAUCAAAAUUGUGAACAUGCAGAUAACUACACCGCAUAUUGUCUGGGAAUAUCUCAUAUGGAGCCUAGCUUUGGUCUAAUCUUACACGACGGGGGCACCACGUUAAAGUUUGUAGAUACACCCGAGAGUUUGUCGGGAUUAUACGUUUUUGUGGUGUAUUUUAACGGGCAUGUUGAAGCCGUAGCAUACACUGUUGUAUCCACAGUAGAUCAUUUUGUAAACGCAAUUGAAGAGCGUGGAUUUCCGCCAACGGCCGGUCAGCCACCGGCGACUACUAAACCCAAGGAAAUUACCCCCGUAAACCCCGGAACGUCACCACUUCUACGAUAUGCCGCAUGGACCGGAGGGCUUGCAGCAGUAGUACUUUUAUGUCUCGUAAUAUUUUUAAUCUGUACGGCUAAACGAAUGAGGGUUAAAGCCUAUAGGGUAGACAAGUGAUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC VZV-GE- delete-574-Y569A(SEQ ID NO: 6)TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGGGACAGTTAATAAACCTGTGGTGGGGGTATTGATGGGGTTCGGAATTATCACGGGAACGTTGCGTATAACGAATCCGGTCAGAGCATCCGTCTTGCGATACGATGATTTTCACATCGATGAAGACAAACTGGATACAAACTCCGTATATGAGCCTTACTACCATTCAGATCATGCGGAGTCTTCATGGGTAAATCGGGGAGAGTCTTCGCGAAAAGCGTACGATCATAACTCACCTTATATATGGCCACGTAATGATTATGATGGATTTTTAGAGAACGCACACGAACACCATGGGGTGTATAATCAGGGCCGTGGTATCGATAGCGGGGAACGGTTAATGCAACCCACACAAATGTCTGCACAGGAGGATCTTGGGGACGATACGGGCATCCACGTTATCCCTACGTTAAACGGCGATGACAGACATAAAATTGTAAATGTGGACCAACGTCAATACGGTGACGTGTTTAAAGGAGATCTTAATCCAAAACCCCAAGGCCAAAGACTCATTGAGGTGTCAGTGGAAGAAAATCACCCGTTTACTTTACGCGCACCGATTCAGCGGATTTATGGAGTCCGGTACACCGAGACTTGGAGCTTTTTGCCGTCATTAACCTGTACGGGAGACGCAGCGCCCGCCATCCAGCATATATGTTTAAAACATACAACATGCTTTCAAGACGTGGTGGTGGATGTGGATTGCGCGGAAAATACTAAAGAGGATCAGTTGGCCGAAATCAGTTACCGTTTTCAAGGTAAGAAGGAAGCGGACCAACCGTGGATTGTTGTAAACACGAGCACACTGTTTGATGAACTCGAATTAGACCCCCCCGAGATTGAACCGGGTGTCTTGAAAGTACTTCGGACAGAAAAACAATACTTGGGTGTGTACATTTGGAACATGCGCGGCTCCGATGGTACGTCTACCTACGCCACGTTTTTGGTCACCTGGAAAGGGGATGAAAAAACAAGAAACCCTACGCCCGCAGTAACTCCTCAACCAAGAGGGGCTGAGTTTCATATGTGGAATTACCACTCGCATGTATTTTCAGTTGGTGATACGTTTAGCTTGGCAATGCATCTTCAGTATAAGATACATGAAGCGCCATTTGATTTGCTGTTAGAGTGGTTGTATGTCCCCATCGATCCTACATGTCAACCAATGCGGTTATATTCTACGTGTTTGTATCATCCCAACGCACCCCAATGCCTCTCTCATATGAATTCCGGTTGTACATTTACCTCGCCACATTTAGCCCAGCGTGTTGCAAGCACAGTGTATCAAAATTGTGAACATGCAGATAACTACACCGCATATTGTCTGGGAATATCTCATATGGAGCCTAGCTTTGGTCTAATCTTACACGACGGGGGCACCACGTTAAAGTTTGTAGATACACCCGAGAGTTTGTCGGGATTATACGTTTTTGTGGTGTATTTTAACGGGCATGTTGAAGCCGTAGCATACACTGTTGTATCCACAGTAGATCATTTTGTAAACGCAATTGAAGAGCGTGGATTTCCGCCAACGGCCGGTCAGCCACCGGCGACTACTAAACCCAAGGAAATTACCCCCGTAAACCCCGGAACGTCACCACTTCTACGATATGCCGCATGGACCGGAGGGCTTGCAGCAGTAGTACTTTTATGTCTCGTAATATTTTTAATCTGTACGGCTAAACGAATGAGGGTTAAAGCCGCCAGGGTAGACAAGTGATGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC VZV-GE- delete-574-Y569A(mRNA)(SEQ ID NO: 128)UCAAGCUUUUGGACCCUCGUACAGAAGCUAAUACGACUCACUAUAGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGGGGACAGUUAAUAAACCUGUGGUGGGGGUAUUGAUGGGGUUCGGAAUUAUCACGGGAACGUUGCGUAUAACGAAUCCGGUCAGAGCAUCCGUCUUGCGAUACGAUGAUUUUCACAUCGAUGAAGACAAACUGGAUACAAACUCCGUAUAUGAGCCUUACUACCAUUCAGAUCAUGCGGAGUCUUCAUGGGUAAAUCGGGGAGAGUCUUCGCGAAAAGCGUACGAUCAUAACUCACCUUAUAUAUGGCCACGUAAUGAUUAUGAUGGAUUUUUAGAGAACGCACACGAACACCAUGGGGUGUAUAAUCAGGGCCGUGGUAUCGAUAGCGGGGAACGGUUAAUGCAACCCACACAAAUGUCUGCACAGGAGGAUCUUGGGGACGAUACGGGCAUCCACGUUAUCCCUACGUUAAACGGCGAUGACAGACAUAAAAUUGUAAAUGUGGACCAACGUCAAUACGGUGACGUGUUUAAAGGAGAUCUUAAUCCAAAACCCCAAGGCCAAAGACUCAUUGAGGUGUCAGUGGAAGAAAAUCACCCGUUUACUUUACGCGCACCGAUUCAGCGGAUUUAUGGAGUCCGGUACACCGAGACUUGGAGCUUUUUGCCGUCAUUAACCUGUACGGGAGACGCAGCGCCCGCCAUCCAGCAUAUAUGUUUAAAACAUACAACAUGCUUUCAAGACGUGGUGGUGGAUGUGGAUUGCGCGGAAAAUACUAAAGAGGAUCAGUUGGCCGAAAUCAGUUACCGUUUUCAAGGUAAGAAGGAAGCGGACCAACCGUGGAUUGUUGUAAACACGAGCACACUGUUUGAUGAACUCGAAUUAGACCCCCCCGAGAUUGAACCGGGUGUCUUGAAAGUACUUCGGACAGAAAAACAAUACUUGGGUGUGUACAUUUGGAACAUGCGCGGCUCCGAUGGUACGUCUACCUACGCCACGUUUUUGGUCACCUGGAAAGGGGAUGAAAAAACAAGAAACCCUACGCCCGCAGUAACUCCUCAACCAAGAGGGGCUGAGUUUCAUAUGUGGAAUUACCACUCGCAUGUAUUUUCAGUUGGUGAUACGUUUAGCUUGGCAAUGCAUCUUCAGUAUAAGAUACAUGAAGCGCCAUUUGAUUUGCUGUUAGAGUGGUUGUAUGUCCCCAUCGAUCCUACAUGUCAACCAAUGCGGUUAUAUUCUACGUGUUUGUAUCAUCCCAACGCACCCCAAUGCCUCUCUCAUAUGAAUUCCGGUUGUACAUUUACCUCGCCACAUUUAGCCCAGCGUGUUGCAAGCACAGUGUAUCAAAAUUGUGAACAUGCAGAUAACUACACCGCAUAUUGUCUGGGAAUAUCUCAUAUGGAGCCUAGCUUUGGUCUAAUCUUACACGACGGGGGCACCACGUUAAAGUUUGUAGAUACACCCGAGAGUUUGUCGGGAUUAUACGUUUUUGUGGUGUAUUUUAACGGGCAUGUUGAAGCCGUAGCAUACACUGUUGUAUCCACAGUAGAUCAUUUUGUAAACGCAAUUGAAGAGCGUGGAUUUCCGCCAACGGCCGGUCAGCCACCGGCGACUACUAAACCCAAGGAAAUUACCCCCGUAAACCCCGGAACGUCACCACUUCUACGAUAUGCCGCAUGGACCGGAGGGCUUGCAGCAGUAGUACUUUUAUGUCUCGUAAUAUUUUUAAUCUGUACGGCUAAACGAAUGAGGGUUAAAGCCGCCAGGGUAGACAAGUGAUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGCVZV-gE-full length-AEAADA (SEQ ID NO: 58) (SEQ ID NO: 7)TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGGGACAGTTAATAAACCTGTGGTGGGGGTATTGATGGGGTTCGGAATTATCACGGGAACGTTGCGTATAACGAATCCGGTCAGAGCATCCGTCTTGCGATACGATGATTTTCACATCGATGAAGACAAACTGGATACAAACTCCGTATATGAGCCTTACTACCATTCAGATCATGCGGAGTCTTCATGGGTAAATCGGGGAGAGTCTTCGCGAAAAGCGTACGATCATAACTCACCTTATATATGGCCACGTAATGATTATGATGGATTTTTAGAGAACGCACACGAACACCATGGGGTGTATAATCAGGGCCGTGGTATCGATAGCGGGGAACGGTTAATGCAACCCACACAAATGTCTGCACAGGAGGATCTTGGGGACGATACGGGCATCCACGTTATCCCTACGTTAAACGGCGATGACAGACATAAAATTGTAAATGTGGACCAACGTCAATACGGTGACGTGTTTAAAGGAGATCTTAATCCAAAACCCCAAGGCCAAAGACTCATTGAGGTGTCAGTGGAAGAAAATCACCCGTTTACTTTACGCGCACCGATTCAGCGGATTTATGGAGTCCGGTACACCGAGACTTGGAGCTTTTTGCCGTCATTAACCTGTACGGGAGACGCAGCGCCCGCCATCCAGCATATATGTTTAAAACATACAACATGCTTTCAAGACGTGGTGGTGGATGTGGATTGCGCGGAAAATACTAAAGAGGATCAGTTGGCCGAAATCAGTTACCGTTTTCAAGGTAAGAAGGAAGCGGACCAACCGTGGATTGTTGTAAACACGAGCACACTGTTTGATGAACTCGAATTAGACCCCCCCGAGATTGAACCGGGTGTCTTGAAAGTACTTCGGACAGAAAAACAATACTTGGGTGTGTACATTTGGAACATGCGCGGCTCCGATGGTACGTCTACCTACGCCACGTTTTTGGTCACCTGGAAAGGGGATGAAAAAACAAGAAACCCTACGCCCGCAGTAACTCCTCAACCAAGAGGGGCTGAGTTTCATATGTGGAATTACCACTCGCATGTATTTTCAGTTGGTGATACGTTTAGCTTGGCAATGCATCTTCAGTATAAGATACATGAAGCGCCATTTGATTTGCTGTTAGAGTGGTTGTATGTCCCCATCGATCCTACATGTCAACCAATGCGGTTATATTCTACGTGTTTGTATCATCCCAACGCACCCCAATGCCTCTCTCATATGAATTCCGGTTGTACATTTACCTCGCCACATTTAGCCCAGCGTGTTGCAAGCACAGTGTATCAAAATTGTGAACATGCAGATAACTACACCGCATATTGTCTGGGAATATCTCATATGGAGCCTAGCTTTGGTCTAATCTTACACGACGGGGGCACCACGTTAAAGTTTGTAGATACACCCGAGAGTTTGTCGGGATTATACGTTTTTGTGGTGTATTTTAACGGGCATGTTGAAGCCGTAGCATACACTGTTGTATCCACAGTAGATCATTTTGTAAACGCAATTGAAGAGCGTGGATTTCCGCCAACGGCCGGTCAGCCACCGGCGACTACTAAACCCAAGGAAATTACCCCCGTAAACCCCGGAACGTCACCACTTCTACGATATGCCGCATGGACCGGAGGGCTTGCAGCAGTAGTACTTTTATGTCTCGTAATATTTTTAATCTGTACGGCTAAACGAATGAGGGTTAAAGCCTATAGGGTAGACAAGTCCCCGTATAACCAAAGCATGTATTACGCTGGCCTTCCAGTGGACGATTTCGAGGACGCCGAAGCCGCCGATGCCGAAGAAGAGTTTGGTAACGCGATTGGAGGGAGTCACGGGGGTTCGAGTTACACGGTGTATATAGATAAGACCCGGTGATGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCVZV-gE-full length-AEAADA (SEQ ID NO: 58)(mRNA) (SEQ ID NO: 129)UCAAGCUUUUGGACCCUCGUACAGAAGCUAAUACGACUCACUAUAGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGGGGACAGUUAAUAAACCUGUGGUGGGGGUAUUGAUGGGGUUCGGAAUUAUCACGGGAACGUUGCGUAUAACGAAUCCGGUCAGAGCAUCCGUCUUGCGAUACGAUGAUUUUCACAUCGAUGAAGACAAACUGGAUACAAACUCCGUAUAUGAGCCUUACUACCAUUCAGAUCAUGCGGAGUCUUCAUGGGUAAAUCGGGGAGAGUCUUCGCGAAAAGCGUACGAUCAUAACUCACCUUAUAUAUGGCCACGUAAUGAUUAUGAUGGAUUUUUAGAGAACGCACACGAACACCAUGGGGUGUAUAAUCAGGGCCGUGGUAUCGAUAGCGGGGAACGGUUAAUGCAACCCACACAAAUGUCUGCACAGGAGGAUCUUGGGGACGAUACGGGCAUCCACGUUAUCCCUACGUUAAACGGCGAUGACAGACAUAAAAUUGUAAAUGUGGACCAACGUCAAUACGGUGACGUGUUUAAAGGAGAUCUUAAUCCAAAACCCCAAGGCCAAAGACUCAUUGAGGUGUCAGUGGAAGAAAAUCACCCGUUUACUUUACGCGCACCGAUUCAGCGGAUUUAUGGAGUCCGGUACACCGAGACUUGGAGCUUUUUGCCGUCAUUAACCUGUACGGGAGACGCAGCGCCCGCCAUCCAGCAUAUAUGUUUAAAACAUACAACAUGCUUUCAAGACGUGGUGGUGGAUGUGGAUUGCGCGGAAAAUACUAAAGAGGAUCAGUUGGCCGAAAUCAGUUACCGUUUUCAAGGUAAGAAGGAAGCGGACCAACCGUGGAUUGUUGUAAACACGAGCACACUGUUUGAUGAACUCGAAUUAGACCCCCCCGAGAUUGAACCGGGUGUCUUGAAAGUACUUCGGACAGAAAAACAAUACUUGGGUGUGUACAUUUGGAACAUGCGCGGCUCCGAUGGUACGUCUACCUACGCCACGUUUUUGGUCACCUGGAAAGGGGAUGAAAAAACAAGAAACCCUACGCCCGCAGUAACUCCUCAACCAAGAGGGGCUGAGUUUCAUAUGUGGAAUUACCACUCGCAUGUAUUUUCAGUUGGUGAUACGUUUAGCUUGGCAAUGCAUCUUCAGUAUAAGAUACAUGAAGCGCCAUUUGAUUUGCUGUUAGAGUGGUUGUAUGUCCCCAUCGAUCCUACAUGUCAACCAAUGCGGUUAUAUUCUACGUGUUUGUAUCAUCCCAACGCACCCCAAUGCCUCUCUCAUAUGAAUUCCGGUUGUACAUUUACCUCGCCACAUUUAGCCCAGCGUGUUGCAAGCACAGUGUAUCAAAAUUGUGAACAUGCAGAUAACUACACCGCAUAUUGUCUGGGAAUAUCUCAUAUGGAGCCUAGCUUUGGUCUAAUCUUACACGACGGGGGCACCACGUUAAAGUUUGUAGAUACACCCGAGAGUUUGUCGGGAUUAUACGUUUUUGUGGUGUAUUUUAACGGGCAUGUUGAAGCCGUAGCAUACACUGUUGUAUCCACAGUAGAUCAUUUUGUAAACGCAAUUGAAGAGCGUGGAUUUCCGCCAACGGCCGGUCAGCCACCGGCGACUACUAAACCCAAGGAAAUUACCCCCGUAAACCCCGGAACGUCACCACUUCUACGAUAUGCCGCAUGGACCGGAGGGCUUGCAGCAGUAGUACUUUUAUGUCUCGUAAUAUUUUUAAUCUGUACGGCUAAACGAAUGAGGGUUAAAGCCUAUAGGGUAGACAAGUCCCCGUAUAACCAAAGCAUGUAUUACGCUGGCCUUCCAGUGGACGAUUUCGAGGACGCCGAAGCCGCCGAUGCCGAAGAAGAGUUUGGUAACGCGAUUGGAGGGAGUCACGGGGGUUCGAGUUACACGGUGUAUAUAGAUAAGACCCGGUGAUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGCVZV-GE-full-AEAADA (SEQ ID NO: 58)-Y582G (SEQ ID NO: 8)TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGGGACAGTTAATAAACCTGTGGTGGGGGTATTGATGGGGTTCGGAATTATCACGGGAACGTTGCGTATAACGAATCCGGTCAGAGCATCCGTCTTGCGATACGATGATTTTCACATCGATGAAGACAAACTGGATACAAACTCCGTATATGAGCCTTACTACCATTCAGATCATGCGGAGTCTTCATGGGTAAATCGGGGAGAGTCTTCGCGAAAAGCGTACGATCATAACTCACCTTATATATGGCCACGTAATGATTATGATGGATTTTTAGAGAACGCACACGAACACCATGGGGTGTATAATCAGGGCCGTGGTATCGATAGCGGGGAACGGTTAATGCAACCCACACAAATGTCTGCACAGGAGGATCTTGGGGACGATACGGGCATCCACGTTATCCCTACGTTAAACGGCGATGACAGACATAAAATTGTAAATGTGGACCAACGTCAATACGGTGACGTGTTTAAAGGAGATCTTAATCCAAAACCCCAAGGCCAAAGACTCATTGAGGTGTCAGTGGAAGAAAATCACCCGTTTACTTTACGCGCACCGATTCAGCGGATTTATGGAGTCCGGTACACCGAGACTTGGAGCTTTTTGCCGTCATTAACCTGTACGGGAGACGCAGCGCCCGCCATCCAGCATATATGTTTAAAACATACAACATGCTTTCAAGACGTGGTGGTGGATGTGGATTGCGCGGAAAATACTAAAGAGGATCAGTTGGCCGAAATCAGTTACCGTTTTCAAGGTAAGAAGGAAGCGGACCAACCGTGGATTGTTGTAAACACGAGCACACTGTTTGATGAACTCGAATTAGACCCCCCCGAGATTGAACCGGGTGTCTTGAAAGTACTTCGGACAGAAAAACAATACTTGGGTGTGTACATTTGGAACATGCGCGGCTCCGATGGTACGTCTACCTACGCCACGTTTTTGGTCACCTGGAAAGGGGATGAAAAAACAAGAAACCCTACGCCCGCAGTAACTCCTCAACCAAGAGGGGCTGAGTTTCATATGTGGAATTACCACTCGCATGTATTTTCAGTTGGTGATACGTTTAGCTTGGCAATGCATCTTCAGTATAAGATACATGAAGCGCCATTTGATTTGCTGTTAGAGTGGTTGTATGTCCCCATCGATCCTACATGTCAACCAATGCGGTTATATTCTACGTGTTTGTATCATCCCAACGCACCCCAATGCCTCTCTCATATGAATTCCGGTTGTACATTTACCTCGCCACATTTAGCCCAGCGTGTTGCAAGCACAGTGTATCAAAATTGTGAACATGCAGATAACTACACCGCATATTGTCTGGGAATATCTCATATGGAGCCTAGCTTTGGTCTAATCTTACACGACGGGGGCACCACGTTAAAGTTTGTAGATACACCCGAGAGTTTGTCGGGATTATACGTTTTTGTGGTGTATTTTAACGGGCATGTTGAAGCCGTAGCATACACTGTTGTATCCACAGTAGATCATTTTGTAAACGCAATTGAAGAGCGTGGATTTCCGCCAACGGCCGGTCAGCCACCGGCGACTACTAAACCCAAGGAAATTACCCCCGTAAACCCCGGAACGTCACCACTTCTACGATATGCCGCATGGACCGGAGGGCTTGCAGCAGTAGTACTTTTATGTCTCGTAATATTTTTAATCTGTACGGCTAAACGAATGAGGGTTAAAGCCTATAGGGTAGACAAGTCCCCGTATAACCAAAGCATGTATGGCGCTGGCCTTCCAGTGGACGATTTCGAGGACGCCGAAGCCGCCGATGCCGAAGAAGAGTTTGGTAACGCGATTGGAGGGAGTCACGGGGGTTCGAGTTACACGGTGTATATAGATAAGACCCGGTGATGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGCVZV-GE-full-AEAADA (SEQ ID NO: 58)-Y582G(mRNA) (SEQ ID NO: 130)UCAAGCUUUUGGACCCUCGUACAGAAGCUAAUACGACUCACUAUAGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGGGGACAGUUAAUAAACCUGUGGUGGGGGUAUUGAUGGGGUUCGGAAUUAUCACGGGAACGUUGCGUAUAACGAAUCCGGUCAGAGCAUCCGUCUUGCGAUACGAUGAUUUUCACAUCGAUGAAGACAAACUGGAUACAAACUCCGUAUAUGAGCCUUACUACCAUUCAGAUCAUGCGGAGUCUUCAUGGGUAAAUCGGGGAGAGUCUUCGCGAAAAGCGUACGAUCAUAACUCACCUUAUAUAUGGCCACGUAAUGAUUAUGAUGGAUUUUUAGAGAACGCACACGAACACCAUGGGGUGUAUAAUCAGGGCCGUGGUAUCGAUAGCGGGGAACGGUUAAUGCAACCCACACAAAUGUCUGCACAGGAGGAUCUUGGGGACGAUACGGGCAUCCACGUUAUCCCUACGUUAAACGGCGAUGACAGACAUAAAAUUGUAAAUGUGGACCAACGUCAAUACGGUGACGUGUUUAAAGGAGAUCUUAAUCCAAAACCCCAAGGCCAAAGACUCAUUGAGGUGUCAGUGGAAGAAAAUCACCCGUUUACUUUACGCGCACCGAUUCAGCGGAUUUAUGGAGUCCGGUACACCGAGACUUGGAGCUUUUUGCCGUCAUUAACCUGUACGGGAGACGCAGCGCCCGCCAUCCAGCAUAUAUGUUUAAAACAUACAACAUGCUUUCAAGACGUGGUGGUGGAUGUGGAUUGCGCGGAAAAUACUAAAGAGGAUCAGUUGGCCGAAAUCAGUUACCGUUUUCAAGGUAAGAAGGAAGCGGACCAACCGUGGAUUGUUGUAAACACGAGCACACUGUUUGAUGAACUCGAAUUAGACCCCCCCGAGAUUGAACCGGGUGUCUUGAAAGUACUUCGGACAGAAAAACAAUACUUGGGUGUGUACAUUUGGAACAUGCGCGGCUCCGAUGGUACGUCUACCUACGCCACGUUUUUGGUCACCUGGAAAGGGGAUGAAAAAACAAGAAACCCUACGCCCGCAGUAACUCCUCAACCAAGAGGGGCUGAGUUUCAUAUGUGGAAUUACCACUCGCAUGUAUUUUCAGUUGGUGAUACGUUUAGCUUGGCAAUGCAUCUUCAGUAUAAGAUACAUGAAGCGCCAUUUGAUUUGCUGUUAGAGUGGUUGUAUGUCCCCAUCGAUCCUACAUGUCAACCAAUGCGGUUAUAUUCUACGUGUUUGUAUCAUCCCAACGCACCCCAAUGCCUCUCUCAUAUGAAUUCCGGUUGUACAUUUACCUCGCCACAUUUAGCCCAGCGUGUUGCAAGCACAGUGUAUCAAAAUUGUGAACAUGCAGAUAACUACACCGCAUAUUGUCUGGGAAUAUCUCAUAUGGAGCCUAGCUUUGGUCUAAUCUUACACGACGGGGGCACCACGUUAAAGUUUGUAGAUACACCCGAGAGUUUGUCGGGAUUAUACGUUUUUGUGGUGUAUUUUAACGGGCAUGUUGAAGCCGUAGCAUACACUGUUGUAUCCACAGUAGAUCAUUUUGUAAACGCAAUUGAAGAGCGUGGAUUUCCGCCAACGGCCGGUCAGCCACCGGCGACUACUAAACCCAAGGAAAUUACCCCCGUAAACCCCGGAACGUCACCACUUCUACGAUAUGCCGCAUGGACCGGAGGGCUUGCAGCAGUAGUACUUUUAUGUCUCGUAAUAUUUUUAAUCUGUACGGCUAAACGAAUGAGGGUUAAAGCCUAUAGGGUAGACAAGUCCCCGUAUAACCAAAGCAUGUAUGGCGCUGGCCUUCCAGUGGACGAUUUCGAGGACGCCGAAGCCGCCGAUGCCGAAGAAGAGUUUGGUAACGCGAUUGGAGGGAGUCACGGGGGUUCGAGUUACACGGUGUAUAUAGAUAAGACCCGGUGAUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGCVZV_gE_Oka_hIgkappa (SEQ ID NO: 41)TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTATAGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAGAGCCACCATGGAGACTCCCGCTCAGCTACTGTTCCTCCTGCTCCTTTGGCTGCCTGATACTACAGGCTCTGTTTTGCGGTACGACGACTTTCACATCGATGAGGACAAGCTCGACACTAATAGCGTGTATGAGCCCTACTACCATTCAGATCACGCCGAGTCCTCTTGGGTGAACAGGGGTGAAAGTTCTAGGAAAGCCTATGATCACAACAGCCCTTATATTTGGCCACGGAATGATTACGACGGATTTCTCGAAAATGCCCACGAGCATCACGGAGTGTACAACCAGGGCCGTGGAATCGACTCTGGGGAGAGATTGATGCAACCTACACAGATGAGCGCCCAGGAAGATCTCGGGGATGATACAGGAATTCACGTTATCCCTACATTAAACGGAGATGACCGCCACAAAATCGTCAATGTCGATCAAAGACAGTATGGAGATGTGTTCAAAGGCGATCTCAACCCTAAGCCGCAGGGCCAGAGACTCATTGAGGTGTCTGTCGAAGAGAACCACCCTTTCACTCTGCGCGCTCCCATTCAGAGAATCTATGGAGTTCGCTATACGGAGACTTGGTCATTCCTTCCTTCCCTGACATGCACCGGAGACGCCGCCCCTGCCATTCAGCACATATGCCTGAAACATACCACCTGTTTCCAGGATGTGGTGGTTGATGTTGATTGTGCTGAAAATACCAAGGAAGACCAACTGGCCGAGATTAGTTACCGGTTCCAAGGGAAAAAGGAAGCCGACCAGCCATGGATTGTGGTTAATACAAGCACTCTGTTCGATGAGCTCGAGCTGGATCCCCCCGAGATAGAACCCGGAGTTCTGAAAGTGCTCCGGACAGAAAAACAATATCTGGGAGTCTACATATGGAACATGCGCGGTTCCGATGGGACCTCCACTTATGCAACCTTTCTCGTCACGTGGAAGGGAGATGAGAAAACTAGGAATCCCACACCCGCTGTCACACCACAGCCAAGAGGGGCTGAGTTCCATATGTGGAACTATCATAGTCACGTGTTTAGTGTCGGAGATACGTTTTCATTGGCTATGCATCTCCAGTACAAGATTCATGAGGCTCCCTTCGATCTGTTGCTTGAGTGGTTGTACGTCCCGATTGACCCGACCTGCCAGCCCATGCGACTGTACAGCACCTGTCTCTACCATCCAAACGCTCCGCAATGTCTGAGCCACATGAACTCTGGGTGTACTTTCACCAGTCCCCACCTCGCCCAGCGGGTGGCCTCTACTGTTTACCAGAACTGTGAGCACGCCGACAACTACACCGCATACTGCCTCGGTATTTCTCACATGGAACCCTCCTTCGGACTCATCCTGCACGATGGGGGCACTACCCTGAAGTTCGTTGATACGCCAGAATCTCTGTCTGGGCTCTATGTTTTCGTGGTCTACTTCAATGGCCATGTCGAGGCCGTGGCCTATACTGTCGTTTCTACCGTGGATCATTTTGTGAACGCCATCGAAGAACGGGGATTCCCCCCTACGGCAGGCCAGCCGCCTGCAACCACCAAGCCCAAGGAAATAACACCAGTGAACCCTGGCACCTCACCTCTCCTAAGATATGCCGCGTGGACAGGGGGACTGGCGGCAGTGGTGCTCCTCTGTCTCGTGATCTTTCTGATCTGTACAGCCAAGAGGATGAGGGTCAAGGCTTATAGAGTGGACAAGTCCCCCTACAATCAGTCAATGTACTACGCCGGCCTTCCCGTTGATGATTTTGAGGATTCCGAGTCCACAGATACTGAGGAAGAGTTCGGTAACGCTATAGGCGGCTCTCACGGGGGTTCAAGCTACACGGTTTACATTGACAAGACACGCTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGGC VZV_gE_Oka_hIgkappa (mRNA) (SEQ ID NO: 131)UCAAGCUUUUGGACCCUCGUACAGAAGCUAAUACGACUCACUAUAGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACCAUGGAGACUCCCGCUCAGCUACUGUUCCUCCUGCUCCUUUGGCUGCCUGAUACUACAGGCUCUGUUUUGCGGUACGACGACUUUCACAUCGAUGAGGACAAGCUCGACACUAAUAGCGUGUAUGAGCCCUACUACCAUUCAGAUCACGCCGAGUCCUCUUGGGUGAACAGGGGUGAAAGUUCUAGGAAAGCCUAUGAUCACAACAGCCCUUAUAUUUGGCCACGGAAUGAUUACGACGGAUUUCUCGAAAAUGCCCACGAGCAUCACGGAGUGUACAACCAGGGCCGUGGAAUCGACUCUGGGGAGAGAUUGAUGCAACCUACACAGAUGAGCGCCCAGGAAGAUCUCGGGGAUGAUACAGGAAUUCACGUUAUCCCUACAUUAAACGGAGAUGACCGCCACAAAAUCGUCAAUGUCGAUCAAAGACAGUAUGGAGAUGUGUUCAAAGGCGAUCUCAACCCUAAGCCGCAGGGCCAGAGACUCAUUGAGGUGUCUGUCGAAGAGAACCACCCUUUCACUCUGCGCGCUCCCAUUCAGAGAAUCUAUGGAGUUCGCUAUACGGAGACUUGGUCAUUCCUUCCUUCCCUGACAUGCACCGGAGACGCCGCCCCUGCCAUUCAGCACAUAUGCCUGAAACAUACCACCUGUUUCCAGGAUGUGGUGGUUGAUGUUGAUUGUGCUGAAAAUACCAAGGAAGACCAACUGGCCGAGAUUAGUUACCGGUUCCAAGGGAAAAAGGAAGCCGACCAGCCAUGGAUUGUGGUUAAUACAAGCACUCUGUUCGAUGAGCUCGAGCUGGAUCCCCCCGAGAUAGAACCCGGAGUUCUGAAAGUGCUCCGGACAGAAAAACAAUAUCUGGGAGUCUACAUAUGGAACAUGCGCGGUUCCGAUGGGACCUCCACUUAUGCAACCUUUCUCGUCACGUGGAAGGGAGAUGAGAAAACUAGGAAUCCCACACCCGCUGUCACACCACAGCCAAGAGGGGCUGAGUUCCAUAUGUGGAACUAUCAUAGUCACGUGUUUAGUGUCGGAGAUACGUUUUCAUUGGCUAUGCAUCUCCAGUACAAGAUUCAUGAGGCUCCCUUCGAUCUGUUGCUUGAGUGGUUGUACGUCCCGAUUGACCCGACCUGCCAGCCCAUGCGACUGUACAGCACCUGUCUCUACCAUCCAAACGCUCCGCAAUGUCUGAGCCACAUGAACUCUGGGUGUACUUUCACCAGUCCCCACCUCGCCCAGCGGGUGGCCUCUACUGUUUACCAGAACUGUGAGCACGCCGACAACUACACCGCAUACUGCCUCGGUAUUUCUCACAUGGAACCCUCCUUCGGACUCAUCCUGCACGAUGGGGGCACUACCCUGAAGUUCGUUGAUACGCCAGAAUCUCUGUCUGGGCUCUAUGUUUUCGUGGUCUACUUCAAUGGCCAUGUCGAGGCCGUGGCCUAUACUGUCGUUUCUACCGUGGAUCAUUUUGUGAACGCCAUCGAAGAACGGGGAUUCCCCCCUACGGCAGGCCAGCCGCCUGCAACCACCAAGCCCAAGGAAAUAACACCAGUGAACCCUGGCACCUCACCUCUCCUAAGAUAUGCCGCGUGGACAGGGGGACUGGCGGCAGUGGUGCUCCUCUGUCUCGUGAUCUUUCUGAUCUGUACAGCCAAGAGGAUGAGGGUCAAGGCUUAUAGAGUGGACAAGUCCCCCUACAAUCAGUCAAUGUACUACGCCGGCCUUCCCGUUGAUGAUUUUGAGGAUUCCGAGUCCACAGAUACUGAGGAAGAGUUCGGUAACGCUAUAGGCGGCUCUCACGGGGGUUCAAGCUACACGGUUUACAUUGACAAGACACGCUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC

TABLE 2 Sequences of Variant VZV gE Constructs Sequence, NT(5′mRNA Sequence mRNA UTR, ORF, 3′ (assumes T100 Name(s) UTR)ORF Sequence, AA ORF Sequence, NT tail) SEQ ID NO: 9 SEQ ID NO: 10SEQ ID NO: 11 SEQ ID NO: 12 VZV_gE_ TCAAGCTTTTGG MGTVNKPVVGVLMGFGIATGGGGACAGTGAATAA G*GGGAAATAAG Oka ACCCTCGTACAG ITGTLRITNPVRASVLRGCCGGTTGTGGGCGTGC AGAGAAAAGAAG AAGCTAATACGA YDDFHIDEDKLDTNSVYTTATGGGCTTTGGGATT AGTAAGAAGAAA CTCACTATAGGG EPYYHSDHAESSWVNRGATTACCGGTACATTACG TATAAGAGCCAC AAATAAGAGAGA ESSRKAYDHNSPYIWPRAATTACCAATCCAGTGC CATGGGGACAGT AAAGAAGAGTAA NDYDGFLENAHEHHGVYGCGCCAGTGTGCTGCGT GAATAAGCCGGT GAAGAAATATAA NQGRGIDSGERLMQPTQTACGACGACTTTCACAT TGTGGGCGTGCT GAGCCACCATGG MSAQEDLGDDTGIHVIPTGACGAGGATAAGCTGG TATGGGCTTTGG GGACAGTGAATA TLNGDDRHKIVNVDQRQATACTAACAGCGTGTAC GATTATTACCGG AGCCGGTTGTGG YGDVFKGDLNPKPQGQRGAACCTTATTACCACTC TACATTACGAAT GCGTGCTTATGG LIEVSVEENHPFTLRAPAGATCATGCCGAATCAA TACCAATCCAGT GCTTTGGGATTA IQRIYGVRYTETWSFLPGCTGGGTTAATAGAGGA GCGCGCCAGTGT TTACCGGTACAT SLTCTGDAAPAIQHICLGAAAGCAGCCGAAAAGC GCTGCGTTACGA TACGAATTACCA KHTTCFQDVVVDVDCAECTACGACCACAACTCAC CGACTTTCACAT ATCCAGTGCGCG NTKEDQLAEISYRFQGKCTTATATTTGGCCCAGA TGACGAGGATAA CCAGTGTGCTGC KEADQPWIVVNTSTLFDAACGATTATGACGGTTT GCTGGATACTAA GTTACGACGACT ELELDPPEIEPGVLKVLCCTGGAAAACGCACATG CAGCGTGTACGA TTCACATTGACG RTEKQYLGVYIWNMRGSAACACCATGGAGTCTAC ACCTTATTACCA AGGATAAGCTGG DGTSTYATFLVTWKGDEAACCAAGGCAGGGGAAT CTCAGATCATGC ATACTAACAGCG KTRNPTPAVTPQPRGAECGACAGTGGCGAGCGTC CGAATCAAGCTG TGTACGAACCTT FHMWNYHSHVFSVGDTFTTATGCAGCCAACACAG GGTTAATAGAGG ATTACCACTCAG SLAMHLQYKIHEAPFDLATGTCGGCACAGGAGGA AGAAAGCAGCCG ATCATGCCGAAT LLEWLYVPIDPTCQPMRTCTCGGTGATGACACCG AAAAGCCTACGA CAAGCTGGGTTA LYSTCLYHPNAPQCLSHGCATACACGTGATTCCC CCACAACTCACC ATAGAGGAGAAA MNSGCTFTSPHLAQRVAACATTAAACGGCGACGA TTATATTTGGCC GCAGCCGAAAAG STVYQNCEHADNYTAYCCAGACATAAGATCGTCA CAGAAACGATTA CCTACGACCACA LGISHMEPSFGLILHDGATGTGGATCAGCGTCAG TGACGGTTTCCT ACTCACCTTATA GTTLKFVDTPESLSGLYTATGGGGATGTCTTTAA GGAAAACGCACA TTTGGCCCAGAA VFVVYFNGHVEAVAYTVAGGCGATTTGAATCCAA TGAACACCATGG ACGATTATGACG VSTVDHFVNAIEERGFPAGCCCCAAGGACAGAGA AGTCTACAACCA GTTTCCTGGAAA PTAGQPPATTKPKEITPCTGATCGAGGTCTCTGT AGGCAGGGGAAT ACGCACATGAAC VNPGTSPLLRYAAWTGGAGAAGAAAATCACCCCT CGACAGTGGCGA ACCATGGAGTCT LAAVVLLCLVIFLICTATCACTTTGCGCGCTCCA GCGTCTTATGCA ACAACCAAGGCA KRMRVKAYRVDKSPYNQATCCAGAGGATTTACGG GCCAACACAGAT GGGGAATCGACA SMYYAGLPVDDFEDSESGGTGCGTTATACCGAAA GTCGGCACAGGA GTGGCGAGCGTC TDTEEEFGNAIGGSHGGCTTGGAGTTTCTTGCCG GGATCTCGGTGA TTATGCAGCCAA SSYTVYIDKTRTCACTGACGTGTACGGG TGACACCGGCAT CACAGATGTCGG GGATGCCGCCCCCGCAAACACGTGATTCC CACAGGAGGATC TCCAGCACATCTGTCTG CACATTAAACGG TCGGTGATGACAAAACACACCACATGCTT CGACGACAGACA CCGGCATACACG TCAGGACGTGGTTGTGGTAAGATCGTCAA TGATTCCCACAT ATGTGGATTGCGCGGAA TGTGGATCAGCG TAAACGGCGACGAACACAAAAGAAGACCA TCAGTATGGGGA ACAGACATAAGA ACTCGCCGAAATCAGCTTGTCTTTAAAGG TCGTCAATGTGG ATCGTTTTCAGGGTAAA CGATTTGAATCC ATCAGCGTCAGTAAAGAGGCCGACCAACC AAAGCCCCAAGG ATGGGGATGTCT GTGGATTGTTGTGAATAACAGAGACTGAT TTAAAGGCGATT CGAGCACGCTCTTCGAT CGAGGTCTCTGT TGAATCCAAAGCGAGCTTGAACTCGATCC AGAAGAAAATCA CCCAAGGACAGA CCCGGAAATCGAGCCTGCCCCTTCACTTT GACTGATCGAGG GGGTTCTAAAAGTGTTG GCGCGCTCCAAT TCTCTGTAGAAGAGGACCGAGAAGCAGTA CCAGAGGATTTA AAAATCACCCCT CCTCGGGGTTTATATCTCGGGGTGCGTTA TCACTTTGCGCG GGAATATGAGAGGCTCC TACCGAAACTTG CTCCAATCCAGAGATGGCACCTCTACCTA GAGTTTCTTGCC GGATTTACGGGG CGCAACGTTTCTGGTTAGTCACTGACGTG TGCGTTATACCG CCTGGAAGGGAGACGAG TACGGGGGATGC AAACTTGGAGTTAAGACACGGAATCCAAC CGCCCCCGCAAT TCTTGCCGTCAC GCCCGCTGTGACCCCTCCCAGCACATCTG TGACGTGTACGG AGCCTAGGGGAGCCGAA TCTGAAACACAC GGGATGCCGCCCTTCCACATGTGGAACTA CACATGCTTTCA CCGCAATCCAGC TCACTCCCATGTATTCAGGACGTGGTTGT ACATCTGTCTGA GTGTGGGTGACACTTTC GGATGTGGATTG AACACACCACATAGCCTGGCCATGCACCT CGCGGAAAACAC GCTTTCAGGACG GCAGTATAAGATTCACGAAAAGAAGACCA TGGTTGTGGATG AGGCACCCTTCGACCTC ACTCGCCGAAAT TGGATTGCGCGGCTGCTGGAGTGGTTGTA CAGCTATCGTTT AAAACACAAAAG CGTACCTATTGATCCCATCAGGGTAAAAA AAGACCAACTCG CTTGTCAGCCCATGCGC AGAGGCCGACCA CCGAAATCAGCTCTGTACTCCACTTGCTT ACCGTGGATTGT ATCGTTTTCAGG GTACCACCCCAATGCACTGTGAATACGAG GTAAAAAAGAGG CACAGTGTCTATCACAC CACGCTCTTCGA CCGACCAACCGTATGAACTCCGGGTGTAC TGAGCTTGAACT GGATTGTTGTGA CTTTACTTCACCCCATCCGATCCCCCGGA ATACGAGCACGC TTGCCCAGCGGGTCGCC AATCGAGCCTGG TCTTCGATGAGCAGCACAGTGTATCAGAA GGTTCTAAAAGT TTGAACTCGATC CTGTGAGCATGCTGACAGTTGAGGACCGA CCCCGGAAATCG ACTATACTGCTTATTGC GAAGCAGTACCT AGCCTGGGGTTCCTCGGAATATCCCATAT CGGGGTTTATAT TAAAAGTGTTGA GGAGCCAAGCTTCGGGCCTGGAATATGAG GGACCGAGAAGC TCATACTGCACGATGGT AGGCTCCGATGG AGTACCTCGGGGGGTACGACACTCAAGTT CACCTCTACCTA TTTATATCTGGA CGTGGACACCCCCGAAACGCAACGTTTCT ATATGAGAGGCT GCCTTTCTGGCTTGTAC GGTTACCTGGAA CCGATGGCACCTGTGTTCGTGGTCTACTT GGGAGACGAGAA CTACCTACGCAA CAATGGACATGTGGAGGGACACGGAATCC CGTTTCTGGTTA CAGTGGCTTACACAGTG AACGCCCGCTGT CCTGGAAGGGAGGTTTCGACAGTTGATCA GACCCCTCAGCC ACGAGAAGACAC CTTTGTAAATGCCATTGTAGGGGAGCCGA GGAATCCAACGC AGGAACGCGGCTTCCCG ATTCCACATGTG CCGCTGTGACCCCCTACAGCGGGCCAGCC GAACTATCACTC CTCAGCCTAGGG CCCTGCGACAACAAAACCCATGTATTCAG GAGCCGAATTCC CAAAAGAGATTACGCCC TGTGGGTGACAC ACATGTGGAACTGTTAATCCTGGGACTAG TTTCAGCCTGGC ATCACTCCCATG TCCATTGCTGAGGTATGCATGCACCTGCA TATTCAGTGTGG CCGCCTGGACTGGCGGT GTATAAGATTCA GTGACACTTTCACTGGCGGCCGTGGTACT CGAGGCACCCTT GCCTGGCCATGC TCTGTGTTTAGTCATATCGACCTCCTGCT ACCTGCAGTATA TTCTGATCTGTACCGCT GGAGTGGTTGTA AGATTCACGAGGAAACGTATGCGGGTCAA CGTACCTATTGA CACCCTTCGACC GGCTTACCGTGTTGACATCCCACTTGTCA TCCTGCTGGAGT AGTCTCCTTACAATCAG GCCCATGCGCCT GGTTGTACGTACTCAATGTACTATGCAGG GTACTCCACTTG CTATTGATCCCA ACTCCCTGTTGACGATTCTTGTACCACCC CTTGTCAGCCCA TCGAAGACTCAGAGAGT CAATGCACCACA TGCGCCTGTACTACAGACACAGAAGAAGA GTGTCTATCACA CCACTTGCTTGT ATTCGGAAACGCTATAGCATGAACTCCGG ACCACCCCAATG GTGGCTCTCACGGAGGT GTGTACCTTTAC CACCACAGTGTCAGCTCGTATACAGTGTA TTCACCCCATCT TATCACACATGA CATCGATAAAACCAGATGCCCAGCGGGT ACTCCGGGTGTA CGCCAGCACAGT CCTTTACTTCAC GTATCAGAACTGCCCATCTTGCCC TGAGCATGCTGA AGCGGGTCGCCA CAACTATACTGC GCACAGTGTATCTTATTGCCTCGG AGAACTGTGAGC AATATCCCATAT ATGCTGACAACT GGAGCCAAGCTTATACTGCTTATT CGGGCTCATACT GCCTCGGAATAT GCACGATGGTGG CCCATATGGAGCTACGACACTCAA CAAGCTTCGGGC GTTCGTGGACAC TCATACTGCACG CCCCGAAAGCCTATGGTGGTACGA TTCTGGCTTGTA CACTCAAGTTCG CGTGTTCGTGGT TGGACACCCCCGCTACTTCAATGG AAAGCCTTTCTG ACATGTGGAGGC GCTTGTACGTGT AGTGGCTTACACTCGTGGTCTACT AGTGGTTTCGAC TCAATGGACATG AGTTGATCACTT TGGAGGCAGTGGTGTAAATGCCAT CTTACACAGTGG TGAGGAACGCGG TTTCGACAGTTG CTTCCCGCCTACATCACTTTGTAA AGCGGGCCAGCC ATGCCATTGAGG CCCTGCGACAAC AACGCGGCTTCCAAAACCAAAAGA CGCCTACAGCGG GATTACGCCCGT GCCAGCCCCCTG TAATCCTGGGACCGACAACAAAAC TAGTCCATTGCT CAAAAGAGATTA GAGGTATGCCGC CGCCCGTTAATCCTGGACTGGCGG CTGGGACTAGTC TCTGGCGGCCGT CATTGCTGAGGT GGTACTTCTGTGATGCCGCCTGGA TTTAGTCATATT CTGGCGGTCTGG TCTGATCTGTAC CGGCCGTGGTACCGCTAAACGTAT TTCTGTGTTTAG GCGGGTCAAGGC TCATATTTCTGA TTACCGTGTTGATCTGTACCGCTA CAAGTCTCCTTA AACGTATGCGGG CAATCAGTCAAT TCAAGGCTTACCGTACTATGCAGG GTGTTGACAAGT ACTCCCTGTTGA CTCCTTACAATC CGATTTCGAAGAAGTCAATGTACT CTCAGAGAGTAC ATGCAGGACTCC AGACACAGAAGA CTGTTGACGATTAGAATTCGGAAA TCGAAGACTCAG CGCTATAGGTGG AGAGTACAGACA CTCTCACGGAGGCAGAAGAAGAAT TAGCTCGTATAC TCGGAAACGCTA AGTGTACATCGA TAGGTGGCTCTCTAAAACCAGATG ACGGAGGTAGCT ATAATAGGCTGG CGTATACAGTGT AGCCTCGGTGGCACATCGATAAAA CATGCTTCTTGC CCAGATGATAAT CCCTTGGGCCTC AGGCTGGAGCCTCCCCCAGCCCCT CGGTGGCCATGC CCTCCCCTTCCT TTCTTGCCCCTT GCACCCGTACCCGGGCCTCCCCCC CCGTGGTCTTTG AGCCCCTCCTCC AATAAAGTCTGA CCTTCCTGCACCGTGGGCGGCAAA CGTACCCCCGTG AAAAAAAAAAAA GTCTTTGAATAA AAAAAAAAAAAAAGTCTGAGTGGG AAAAAAAAAAAA CGGC AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAA ATCTAG SEQ ID NO: 13 SEQ ID NO: 14SEQ ID NO: 15 SEQ ID NO: 16 VZV_gE_ TCAAGCTTTTGG METPAQLLFLLLLWLPDATGGAGACTCCCGCTCA G*GGGAAATAAG Oka_hIg ACCCTCGTACAG TTGSVLRYDDFHIDEDKGCTACTGTTCCTCCTGC AGAGAAAAGAAG kappa AAGCTAATACGA LDTNSVYEPYYHSDHAETCCTTTGGCTGCCTGAT AGTAAGAAGAAA CTCACTATAGGG SSWVNRGESSRKAYDHNACTACAGGCTCTGTTTT TATAAGAGCCAC AAATAAGAGAGA SPYIWPRNDYDGFLENAGCGGTACGACGACTTTC CATGGAGACTCC AAAGAAGAGTAA HEHHGVYNQGRGIDSGEACATCGATGAGGACAAG CGCTCAGCTACT GAAGAAATATAA RLMQPTQMSAQEDLGDDCTCGACACTAATAGCGT GTTCCTCCTGCT GAGCCACCATGG TGIHVIPTLNGDDRHKIGTATGAGCCCTACTACC CCTTTGGCTGCC AGACTCCCGCTC VNVDQRQYGDVFKGDLNATTCAGATCACGCCGAG TGATACTACAGG AGCTACTGTTCC PKPQGQRLIEVSVEENHTCCTCTTGGGTGAACAG CTCTGTTTTGCG TCCTGCTCCTTT PFTLRAPIQRIYGVRYTGGGTGAAAGTTCTAGGA GTACGACGACTT GGCTGCCTGATA ETWSFLPSLTCTGDAAPAAGCCTATGATCACAAC TCACATCGATGA CTACAGGCTCTG AIQHICLKHTTCFQDVVAGCCCTTATATTTGGCC GGACAAGCTCGA TTTTGCGGTACG VDVDCAENTKEDQLAEIACGGAATGATTACGACG CACTAATAGCGT ACGACTTTCACA SYRFQGKKEADQPWIVVGATTTCTCGAAAATGCC GTATGAGCCCTA TCGATGAGGACA NTSTLFDELELDPPEIECACGAGCATCACGGAGT CTACCATTCAGA AGCTCGACACTA PGVLKVLRTEKQYLGVYGTACAACCAGGGCCGTG TCACGCCGAGTC ATAGCGTGTATG IWNMRGSDGTSTYATFLGAATCGACTCTGGGGAG CTCTTGGGTGAA AGCCCTACTACC VTWKGDEKTRNPTPAVTAGATTGATGCAACCTAC CAGGGGTGAAAG ATTCAGATCACG PQPRGAEFHMWNYHSHVACAGATGAGCGCCCAGG TTCTAGGAAAGC CCGAGTCCTCTT FSVGDTFSLAMHLQYKIAAGATCTCGGGGATGAT CTATGATCACAA GGGTGAACAGGG HEAPFDLLLEWLYVPIDACAGGAATTCACGTTAT CAGCCCTTATAT GTGAAAGTTCTA PTCQPMRLYSTCLYHPNCCCTACATTAAACGGAG TTGGCCACGGAA GGAAAGCCTATG APQCLSHMNSGCTFTSPATGACCGCCACAAAATC TGATTACGACGG ATCACAACAGCC HLAQRVASTVYQNCEHAGTCAATGTCGATCAAAG ATTTCTCGAAAA CTTATATTTGGC DNYTAYCLGISHMEPSFACAGTATGGAGATGTGT TGCCCACGAGCA CACGGAATGATT GLILHDGGTTLKFVDTPTCAAAGGCGATCTCAAC TCACGGAGTGTA ACGACGGATTTC ESLSGLYVFVVYFNGHVCCTAAGCCGCAGGGCCA CAACCAGGGCCG TCGAAAATGCCC EAVAYTVVSTVDHFVNAGAGACTCATTGAGGTGT TGGAATCGACTC ACGAGCATCACG IEERGFPPTAGQPPATTCTGTCGAAGAGAACCAC TGGGGAGAGATT GAGTGTACAACC KPKEITPVNPGTSPLLRCCTTTCACTCTGCGCGC GATGCAACCTAC AGGGCCGTGGAA YAAWTGGLAAVVLLCLVTCCCATTCAGAGAATCT ACAGATGAGCGC TCGACTCTGGGG IFLICTAKRMRVKAYRVATGGAGTTCGCTATACG CCAGGAAGATCT AGAGATTGATGC DKSPYNQSMYYAGLPVDGAGACTTGGTCATTCCT CGGGGATGATAC AACCTACACAGA DFEDSESTDTEEEFGNATCCTTCCCTGACATGCA AGGAATTCACGT TGAGCGCCCAGG IGGSHGGSSYTVYIDKTCCGGAGACGCCGCCCCT TATCCCTACATT AAGATCTCGGGG R GCCATTCAGCACATATGAAACGGAGATGA ATGATACAGGAA CCTGAAACATACCACCT CCGCCACAAAAT TTCACGTTATCCGTTTCCAGGATGTGGTG CGTCAATGTCGA CTACATTAAACG GTTGATGTTGATTGTGCTCAAAGACAGTA GAGATGACCGCC TGAAAATACCAAGGAAG TGGAGATGTGTT ACAAAATCGTCAACCAACTGGCCGAGATT CAAAGGCGATCT ATGTCGATCAAA AGTTACCGGTTCCAAGGCAACCCTAAGCC GACAGTATGGAG GAAAAAGGAAGCCGACC GCAGGGCCAGAG ATGTGTTCAAAGAGCCATGGATTGTGGTT ACTCATTGAGGT GCGATCTCAACC AATACAAGCACTCTGTTGTCTGTCGAAGA CTAAGCCGCAGG CGATGAGCTCGAGCTGG GAACCACCCTTT GCCAGAGACTCAATCCCCCCGAGATAGAA CACTCTGCGCGC TTGAGGTGTCTG CCCGGAGTTCTGAAAGTTCCCATTCAGAG TCGAAGAGAACC GCTCCGGACAGAAAAAC AATCTATGGAGT ACCCTTTCACTCAATATCTGGGAGTCTAC TCGCTATACGGA TGCGCGCTCCCA ATATGGAACATGCGCGGGACTTGGTCATT TTCAGAGAATCT TTCCGATGGGACCTCCA CCTTCCTTCCCT ATGGAGTTCGCTCTTATGCAACCTTTCTC GACATGCACCGG ATACGGAGACTT GTCACGTGGAAGGGAGAAGACGCCGCCCC GGTCATTCCTTC TGAGAAAACTAGGAATC TGCCATTCAGCA CTTCCCTGACATCCACACCCGCTGTCACA CATATGCCTGAA GCACCGGAGACG CCACAGCCAAGAGGGGCACATACCACCTG CCGCCCCTGCCA TGAGTTCCATATGTGGA TTTCCAGGATGT TTCAGCACATATACTATCATAGTCACGTG GGTGGTTGATGT GCCTGAAACATA TTTAGTGTCGGAGATACTGATTGTGCTGA CCACCTGTTTCC GTTTTCATTGGCTATGC AAATACCAAGGA AGGATGTGGTGGATCTCCAGTACAAGATT AGACCAACTGGC TTGATGTTGATT CATGAGGCTCCCTTCGACGAGATTAGTTA GTGCTGAAAATA TCTGTTGCTTGAGTGGT CCGGTTCCAAGG CCAAGGAAGACCTGTACGTCCCGATTGAC GAAAAAGGAAGC AACTGGCCGAGA CCGACCTGCCAGCCCATCGACCAGCCATG TTAGTTACCGGT GCGACTGTACAGCACCT GATTGTGGTTAA TCCAAGGGAAAAGTCTCTACCATCCAAAC TACAAGCACTCT AGGAAGCCGACC GCTCCGCAATGTCTGAGGTTCGATGAGCT AGCCATGGATTG CCACATGAACTCTGGGT CGAGCTGGATCC TGGTTAATACAAGTACTTTCACCAGTCCC CCCCGAGATAGA GCACTCTGTTCG CACCTCGCCCAGCGGGTACCCGGAGTTCT ATGAGCTCGAGC GGCCTCTACTGTTTACC GAAAGTGCTCCG TGGATCCCCCCGAGAACTGTGAGCACGCC GACAGAAAAACA AGATAGAACCCG GACAACTACACCGCATAATATCTGGGAGT GAGTTCTGAAAG CTGCCTCGGTATTTCTC CTACATATGGAA TGCTCCGGACAGACATGGAACCCTCCTTC CATGCGCGGTTC AAAAACAATATC GGACTCATCCTGCACGACGATGGGACCTC TGGGAGTCTACA TGGGGGCACTACCCTGA CACTTATGCAAC TATGGAACATGCAGTTCGTTGATACGCCA CTTTCTCGTCAC GCGGTTCCGATG GAATCTCTGTCTGGGCTGTGGAAGGGAGA GGACCTCCACTT CTATGTTTTCGTGGTCT TGAGAAAACTAG ATGCAACCTTTCACTTCAATGGCCATGTC GAATCCCACACC TCGTCACGTGGA GAGGCCGTGGCCTATACCGCTGTCACACC AGGGAGATGAGA TGTCGTTTCTACCGTGG ACAGCCAAGAGG AAACTAGGAATCATCATTTTGTGAACGCC GGCTGAGTTCCA CCACACCCGCTG ATCGAAGAACGGGGATTTATGTGGAACTA TCACACCACAGC CCCCCCTACGGCAGGCC TCATAGTCACGT CAAGAGGGGCTGAGCCGCCTGCAACCACC GTTTAGTGTCGG AGTTCCATATGT AAGCCCAAGGAAATAACAGATACGTTTTC GGAACTATCATA ACCAGTGAACCCTGGCA ATTGGCTATGCA GTCACGTGTTTACCTCACCTCTCCTAAGA TCTCCAGTACAA GTGTCGGAGATA TATGCCGCGTGGACAGGGATTCATGAGGC CGTTTTCATTGG GGGACTGGCGGCAGTGG TCCCTTCGATCT CTATGCATCTCCTGCTCCTCTGTCTCGTG GTTGCTTGAGTG AGTACAAGATTC ATCTTTCTGATCTGTACGTTGTACGTCCC ATGAGGCTCCCT AGCCAAGAGGATGAGGG GATTGACCCGAC TCGATCTGTTGCTCAAGGCTTATAGAGTG CTGCCAGCCCAT TTGAGTGGTTGT GACAAGTCCCCCTACAAGCGACTGTACAG ACGTCCCGATTG TCAGTCAATGTACTACG CACCTGTCTCTA ACCCGACCTGCCCCGGCCTTCCCGTTGAT CCATCCAAACGC AGCCCATGCGAC GATTTTGAGGATTCCGATCCGCAATGTCT TGTACAGCACCT GTCCACAGATACTGAGG GAGCCACATGAA GTCTCTACCATCAAGAGTTCGGTAACGCT CTCTGGGTGTAC CAAACGCTCCGC ATAGGCGGCTCTCACGGTTTCACCAGTCC AATGTCTGAGCC GGGTTCAAGCTACACGG CCACCTCGCCCA ACATGAACTCTGTTTACATTGACAAGACA GCGGGTGGCCTC GGTGTACTTTCA CGC TACTGTTTACCACCAGTCCCCACC GAACTGTGAGCA TCGCCCAGCGGG CGCCGACAACTA TGGCCTCTACTGCACCGCATACTG TTTACCAGAACT CCTCGGTATTTC GTGAGCACGCCG TCACATGGAACCACAACTACACCG CTCCTTCGGACT CATACTGCCTCG CATCCTGCACGA GTATTTCTCACATGGGGGCACTAC TGGAACCCTCCT CCTGAAGTTCGT TCGGACTCATCC TGATACGCCAGATGCACGATGGGG ATCTCTGTCTGG GCACTACCCTGA GCTCTATGTTTT AGTTCGTTGATACGTGGTCTACTT CGCCAGAATCTC CAATGGCCATGT TGTCTGGGCTCT CGAGGCCGTGGCATGTTTTCGTGG CTATACTGTCGT TCTACTTCAATG TTCTACCGTGGA GCCATGTCGAGGTCATTTTGTGAA CCGTGGCCTATA CGCCATCGAAGA CTGTCGTTTCTA ACGGGGATTCCCCCGTGGATCATT CCCTACGGCAGG TTGTGAACGCCA CCAGCCGCCTGC TCGAAGAACGGGAACCACCAAGCC GATTCCCCCCTA CAAGGAAATAAC CGGCAGGCCAGC ACCAGTGAACCCCGCCTGCAACCA TGGCACCTCACC CCAAGCCCAAGG TCTCCTAAGATA AAATAACACCAGTGCCGCGTGGAC TGAACCCTGGCA AGGGGGACTGGC CCTCACCTCTCC GGCAGTGGTGCTTAAGATATGCCG CCTCTGTCTCGT CGTGGACAGGGG GATCTTTCTGAT GACTGGCGGCAGCTGTACAGCCAA TGGTGCTCCTCT GAGGATGAGGGT GTCTCGTGATCT CAAGGCTTATAGTTCTGATCTGTA AGTGGACAAGTC CAGCCAAGAGGA CCCCTACAATCA TGAGGGTCAAGGGTCAATGTACTA CTTATAGAGTGG CGCCGGCCTTCC ACAAGTCCCCCT CGTTGATGATTTACAATCAGTCAA TGAGGATTCCGA TGTACTACGCCG GTCCACAGATAC GCCTTCCCGTTGTGAGGAAGAGTT ATGATTTTGAGG CGGTAACGCTAT ATTCCGAGTCCA AGGCGGCTCTCACAGATACTGAGG CGGGGGTTCAAG AAGAGTTCGGTA CTACACGGTTTA ACGCTATAGGCGCATTGACAAGAC GCTCTCACGGGG ACGCTGATAATA GTTCAAGCTACA GGCTGGAGCCTCCGGTTTACATTG GGTGGCCATGCT ACAAGACACGCT TCTTGCCCCTTG GATAATAGGCTGGGCCTCCCCCCA GAGCCTCGGTGG GCCCCTCCTCCC CCATGCTTCTTG CTTCCTGCACCCCCCCTTGGGCCT GTACCCCCGTGG CCCCCCAGCCCC TCTTTGAATAAA TCCTCCCCTTCCGTCTGAGTGGGC TGCACCCGTACC GGCAAAAAAAAA CCCGTGGTCTTT AAAAAAAAAAAAGAATAAAGTCTG AAAAAAAAAAAA AGTGGGCGGC AAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAATCTAG SEQ ID NO: 17SEQ ID NO: 18 SEQ ID NO: 19 SEQ ID NO: 20 VZV-GE- TCAAGCTTTTGGMGTVNKPVVGVLMGFGI ATGGGGACAGTTAATAA G*GGGAAATAAG delete- ACCCTCGTACAGITGTLRITNPVRASVLR ACCTGTGGTGGGGGTAT AGAGAAAAGAAG 562 AAGCTAATACGAYDDFHIDEDKLDTNSVY TGATGGGGTTCGGAATT AGTAAGAAGAAA CTCACTATAGGGEPYYHSDHAESSWVNRG ATCACGGGAACGTTGCG TATAAGAGCCAC AAATAAGAGAGAESSRKAYDHNSPYIWPR TATAACGAATCCGGTCA CATGGGGACAGT AAAGAAGAGTAANDYDGFLENAHEHHGVY GAGCATCCGTCTTGCGA TAATAAACCTGT GAAGAAATATAANQGRGIDSGERLMQPTQ TACGATGATTTTCACAT GGTGGGGGTATT GAGCCACCATGGMSAQEDLGDDTGIHVIP CGATGAAGACAAACTGG GATGGGGTTCGG GGACAGTTAATATLNGDDRHKIVNVDQRQ ATACAAACTCCGTATAT AATTATCACGGG AACCTGTGGTGGYGDVFKGDLNPKPQGQR GAGCCTTACTACCATTC AACGTTGCGTAT GGGTATTGATGGLIEVSVEENHPFTLRAP AGATCATGCGGAGTCTT AACGAATCCGGT GGTTCGGAATTAIQRIYGVRYTETWSFLP CATGGGTAAATCGGGGA CAGAGCATCCGT TCACGGGAACGTSLTCTGDAAPAIQHICL GAGTCTTCGCGAAAAGC CTTGCGATACGA TGCGTATAACGAKHTTCFQDVVVDVDCAE GTACGATCATAACTCAC TGATTTTCACAT ATCCGGTCAGAGNTKEDQLAEISYRFQGK CTTATATATGGCCACGT CGATGAAGACAA CATCCGTCTTGCKEADQPWIVVNTSTLFD AATGATTATGATGGATT ACTGGATACAAA GATACGATGATTELELDPPEIEPGVLKVL TTTAGAGAACGCACACG CTCCGTATATGA TTCACATCGATGRTEKQYLGVYIWNMRGS AACACCATGGGGTGTAT GCCTTACTACCA AAGACAAACTGGDGTSTYATFLVTWKGDE AATCAGGGCCGTGGTAT TTCAGATCATGC ATACAAACTCCGKTRNPTPAVTPQPRGAE CGATAGCGGGGAACGGT GGAGTCTTCATG TATATGAGCCTTFHMWNYHSHVFSVGDTF TAATGCAACCCACACAA GGTAAATCGGGG ACTACCATTCAGSLAMHLQYKIHEAPFDL ATGTCTGCACAGGAGGA AGAGTCTTCGCG ATCATGCGGAGTLLEWLYVPIDPTCQPMR TCTTGGGGACGATACGG AAAAGCGTACGA CTTCATGGGTAALYSTCLYHPNAPQCLSH GCATCCACGTTATCCCT TCATAACTCACC ATCGGGGAGAGTMNSGCTFTSPHLAQRVA ACGTTAAACGGCGATGA TTATATATGGCC CTTCGCGAAAAGSTVYQNCEHADNYTAYC CAGACATAAAATTGTAA ACGTAATGATTA CGTACGATCATALGISHMEPSFGLILHDG ATGTGGACCAACGTCAA TGATGGATTTTT ACTCACCTTATAGTTLKFVDTPESLSGLY TACGGTGACGTGTTTAA AGAGAACGCACA TATGGCCACGTAVFVVYFNGHVEAVAYTV AGGAGATCTTAATCCAA CGAACACCATGG ATGATTATGATGVSTVDHFVNAIEERGFP AACCCCAAGGCCAAAGA GGTGTATAATCA GATTTTTAGAGAPTAGQPPATTKPKEITP CTCATTGAGGTGTCAGT GGGCCGTGGTAT ACGCACACGAACVNPGTSPLLRYAAWTGG GGAAGAAAATCACCCGT CGATAGCGGGGA ACCATGGGGTGTLAAVVLLCLVIFLICTA TTACTTTACGCGCACCG ACGGTTAATGCA ATAATCAGGGCC *ATTCAGCGGATTTATGG ACCCACACAAAT GTGGTATCGATA AGTCCGGTACACCGAGAGTCTGCACAGGA GCGGGGAACGGT CTTGGAGCTTTTTGCCG GGATCTTGGGGA TAATGCAACCCATCATTAACCTGTACGGG CGATACGGGCAT CACAAATGTCTG AGACGCAGCGCCCGCCACCACGTTATCCC CACAGGAGGATC TCCAGCATATATGTTTA TACGTTAAACGG TTGGGGACGATAAAACATACAACATGCTT CGATGACAGACA CGGGCATCCACG TCAAGACGTGGTGGTGGTAAAATTGTAAA TTATCCCTACGT ATGTGGATTGCGCGGAA TGTGGACCAACG TAAACGGCGATGAATACTAAAGAGGATCA TCAATACGGTGA ACAGACATAAAA GTTGGCCGAAATCAGTTCGTGTTTAAAGG TTGTAAATGTGG ACCGTTTTCAAGGTAAG AGATCTTAATCC ACCAACGTCAATAAGGAAGCGGACCAACC AAAACCCCAAGG ACGGTGACGTGT GTGGATTGTTGTAAACACCAAAGACTCAT TTAAAGGAGATC CGAGCACACTGTTTGAT TGAGGTGTCAGT TTAATCCAAAACGAACTCGAATTAGACCC GGAAGAAAATCA CCCAAGGCCAAA CCCCGAGATTGAACCGGCCCGTTTACTTT GACTCATTGAGG GTGTCTTGAAAGTACTT ACGCGCACCGAT TGTCAGTGGAAGCGGACAGAAAAACAATA TCAGCGGATTTA AAAATCACCCGT CTTGGGTGTGTACATTTTGGAGTCCGGTA TTACTTTACGCG GGAACATGCGCGGCTCC CACCGAGACTTG CACCGATTCAGCGATGGTACGTCTACCTA GAGCTTTTTGCC GGATTTATGGAG CGCCACGTTTTTGGTCAGTCATTAACCTG TCCGGTACACCG CCTGGAAAGGGGATGAA TACGGGAGACGC AGACTTGGAGCTAAAACAAGAAACCCTAC AGCGCCCGCCAT TTTTGCCGTCAT GCCCGCAGTAACTCCTCCCAGCATATATG TAACCTGTACGG AACCAAGAGGGGCTGAG TTTAAAACATAC GAGACGCAGCGCTTTCATATGTGGAATTA AACATGCTTTCA CCGCCATCCAGC CCACTCGCATGTATTTTAGACGTGGTGGT ATATATGTTTAA CAGTTGGTGATACGTTT GGATGTGGATTG AACATACAACATAGCTTGGCAATGCATCT CGCGGAAAATAC GCTTTCAAGACG TCAGTATAAGATACATGTAAAGAGGATCA TGGTGGTGGATG AAGCGCCATTTGATTTG GTTGGCCGAAAT TGGATTGCGCGGCTGTTAGAGTGGTTGTA CAGTTACCGTTT AAAATACTAAAG TGTCCCCATCGATCCTATCAAGGTAAGAA AGGATCAGTTGG CATGTCAACCAATGCGG GGAAGCGGACCA CCGAAATCAGTTTTATATTCTACGTGTTT ACCGTGGATTGT ACCGTTTTCAAG GTATCATCCCAACGCACTGTAAACACGAG GTAAGAAGGAAG CCCAATGCCTCTCTCAT CACACTGTTTGA CGGACCAACCGTATGAATTCCGGTTGTAC TGAACTCGAATT GGATTGTTGTAA ATTTACCTCGCCACATTAGACCCCCCCGA ACACGAGCACAC TAGCCCAGCGTGTTGCA GATTGAACCGGG TGTTTGATGAACAGCACAGTGTATCAAAA TGTCTTGAAAGT TCGAATTAGACC TTGTGAACATGCAGATAACTTCGGACAGA CCCCCGAGATTG ACTACACCGCATATTGT AAAACAATACTT AACCGGGTGTCTCTGGGAATATCTCATAT GGGTGTGTACAT TGAAAGTACTTC GGAGCCTAGCTTTGGTCTTGGAACATGCG GGACAGAAAAAC TAATCTTACACGACGGG CGGCTCCGATGG AATACTTGGGTGGGCACCACGTTAAAGTT TACGTCTACCTA TGTACATTTGGA TGTAGATACACCCGAGACGCCACGTTTTT ACATGCGCGGCT GTTTGTCGGGATTATAC GGTCACCTGGAA CCGATGGTACGTGTTTTTGTGGTGTATTT AGGGGATGAAAA CTACCTACGCCA TAACGGGCATGTTGAAGAACAAGAAACCC CGTTTTTGGTCA CCGTAGCATACACTGTT TACGCCCGCAGT CCTGGAAAGGGGGTATCCACAGTAGATCA AACTCCTCAACC ATGAAAAAACAA TTTTGTAAACGCAATTGAAGAGGGGCTGA GAAACCCTACGC AAGAGCGTGGATTTCCG GTTTCATATGTG CCGCAGTAACTCCCAACGGCCGGTCAGCC GAATTACCACTC CTCAACCAAGAG ACCGGCGACTACTAAACGCATGTATTTTC GGGCTGAGTTTC CCAAGGAAATTACCCCC AGTTGGTGATAC ATATGTGGAATTGTAAACCCCGGAACGTC GTTTAGCTTGGC ACCACTCGCATG ACCACTTCTACGATATGAATGCATCTTCA TATTTTCAGTTG CCGCATGGACCGGAGGG GTATAAGATACA GTGATACGTTTACTTGCAGCAGTAGTACT TGAAGCGCCATT GCTTGGCAATGC TTTATGTCTCGTAATATTGATTTGCTGTT ATCTTCAGTATA TTTTAATCTGTACGGCT AGAGTGGTTGTA AGATACATGAAGTGA TGTCCCCATCGA CGCCATTTGATT TCCTACATGTCA TGCTGTTAGAGT ACCAATGCGGTTGGTTGTATGTCC ATATTCTACGTG CCATCGATCCTA TTTGTATCATCC CATGTCAACCAACAACGCACCCCA TGCGGTTATATT ATGCCTCTCTCA CTACGTGTTTGT TATGAATTCCGGATCATCCCAACG TTGTACATTTAC CACCCCAATGCC CTCGCCACATTT TCTCTCATATGAAGCCCAGCGTGT ATTCCGGTTGTA TGCAAGCACAGT CATTTACCTCGC GTATCAAAATTGCACATTTAGCCC TGAACATGCAGA AGCGTGTTGCAA TAACTACACCGC GCACAGTGTATCATATTGTCTGGG AAAATTGTGAAC AATATCTCATAT ATGCAGATAACT GGAGCCTAGCTTACACCGCATATT TGGTCTAATCTT GTCTGGGAATAT ACACGACGGGGG CTCATATGGAGCCACCACGTTAAA CTAGCTTTGGTC GTTTGTAGATAC TAATCTTACACG ACCCGAGAGTTTACGGGGGCACCA GTCGGGATTATA CGTTAAAGTTTG CGTTTTTGTGGT TAGATACACCCGGTATTTTAACGG AGAGTTTGTCGG GCATGTTGAAGC GATTATACGTTT CGTAGCATACACTTGTGGTGTATT TGTTGTATCCAC TTAACGGGCATG AGTAGATCATTT TTGAAGCCGTAGTGTAAACGCAAT CATACACTGTTG TGAAGAGCGTGG TATCCACAGTAG ATTTCCGCCAACATCATTTTGTAA GGCCGGTCAGCC ACGCAATTGAAG ACCGGCGACTAC AGCGTGGATTTCTAAACCCAAGGA CGCCAACGGCCG AATTACCCCCGT GTCAGCCACCGG AAACCCCGGAACCGACTACTAAAC GTCACCACTTCT CCAAGGAAATTA ACGATATGCCGC CCCCCGTAAACCATGGACCGGAGG CCGGAACGTCAC GCTTGCAGCAGT CACTTCTACGAT AGTACTTTTATGATGCCGCATGGA TCTCGTAATATT CCGGAGGGCTTG TTTAATCTGTAC CAGCAGTAGTACGGCTTGATGATA TTTTATGTCTCG ATAGGCTGGAGC TAATATTTTTAA CTCGGTGGCCATTCTGTACGGCTT GCTTCTTGCCCC GATGATAATAGG TTGGGCCTCCCC CTGGAGCCTCGGCCAGCCCCTCCT TGGCCATGCTTC CCCCTTCCTGCA TTGCCCCTTGGG CCCGTACCCCCGCCTCCCCCCAGC TGGTCTTTGAAT CCCTCCTCCCCT AAAGTCTGAGTG TCCTGCACCCGTGGCGGCAAAAAA ACCCCCGTGGTC AAAAAAAAAAAA TTTGAATAAAGT AAAAAAAAAAAACTGAGTGGGCGG AAAAAAAAAAAA C AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAATC TAG SEQ ID NO: 21 SEQ ID NO: 22 SEQ ID NO: 23SEQ ID NO: 24 VZV-GE- TCAAGCTTTTGG METPAQLLFLLLLWLPD ATGGAAACCCCGGCGCAG*GGGAAATAAG delete- ACCCTCGTACAG TTGSVLRYDDFHIDEDK GCTGCTGTTTCTGCTGCAGAGAAAAGAAG 562- AAGCTAATACGA LDTNSVYEPYYHSDHAE TGCTGTGGCTGCCGGATAGTAAGAAGAAA replace CTCACTATAGGG SSWVNRGESSRKAYDHN ACCACCGGCTCCGTCTTTATAAGAGCCAC dSP- AAATAAGAGAGA SPYIWPRNDYDGFLENA GCGATACGATGATTTTCCATGGAAACCCC withIgKappa AAAGAAGAGTAA HEHHGVYNQGRGIDSGEACATCGATGAAGACAAA GGCGCAGCTGCT GAAGAAATATAA RLMQPTQMSAQEDLGDDCTGGATACAAACTCCGT GTTTCTGCTGCT GAGCCACCATGG TGIHVIPTLNGDDRHKIATATGAGCCTTACTACC GCTGTGGCTGCC AAACCCCGGCGC VNVDQRQYGDVFKGDLNATTCAGATCATGCGGAG GGATACCACCGG AGCTGCTGTTTC PKPQGQRLIEVSVEENHTCTTCATGGGTAAATCG CTCCGTCTTGCG TGCTGCTGCTGT PFTLRAPIQRIYGVRYTGGGAGAGTCTTCGCGAA ATACGATGATTT GGCTGCCGGATA ETWSFLPSLTCTGDAAPAAGCGTACGATCATAAC TCACATCGATGA CCACCGGCTCCG AIQHICLKHTTCFQDVVTCACCTTATATATGGCC AGACAAACTGGA TCTTGCGATACG VDVDCAENTKEDQLAEIACGTAATGATTATGATG TACAAACTCCGT ATGATTTTCACA SYRFQGKKEADQPWIVVGATTTTTAGAGAACGCA ATATGAGCCTTA TCGATGAAGACA NTSTLFDELELDPPEIECACGAACACCATGGGGT CTACCATTCAGA AACTGGATACAA PGVLKVLRTEKQYLGVYGTATAATCAGGGCCGTG TCATGCGGAGTC ACTCCGTATATG IWNMRGSDGTSTYATFLGTATCGATAGCGGGGAA TTCATGGGTAAA AGCCTTACTACC VTWKGDEKTRNPTPAVTCGGTTAATGCAACCCAC TCGGGGAGAGTC ATTCAGATCATG PQPRGAEFHMWNYHSHVACAAATGTCTGCACAGG TTCGCGAAAAGC CGGAGTCTTCAT FSVGDTFSLAMHLQYKIAGGATCTTGGGGACGAT GTACGATCATAA GGGTAAATCGGG HEAPFDLLLEWLYVPIDACGGGCATCCACGTTAT CTCACCTTATAT GAGAGTCTTCGC PTCQPMRLYSTCLYHPNCCCTACGTTAAACGGCG ATGGCCACGTAA GAAAAGCGTACG APQCLSHMNSGCTFTSPATGACAGACATAAAATT TGATTATGATGG ATCATAACTCAC HLAQRVASTVYQNCEHAGTAAATGTGGACCAACG ATTTTTAGAGAA CTTATATATGGC DNYTAYCLGISHMEPSFTCAATACGGTGACGTGT CGCACACGAACA CACGTAATGATT GLILHDGGTTLKFVDTPTTAAAGGAGATCTTAAT CCATGGGGTGTA ATGATGGATTTT ESLSGLYVFVVYFNGHVCCAAAACCCCAAGGCCA TAATCAGGGCCG TAGAGAACGCAC EAVAYTVVSTVDHFVNAAAGACTCATTGAGGTGT TGGTATCGATAG ACGAACACCATG IEERGFPPTAGQPPATTCAGTGGAAGAAAATCAC CGGGGAACGGTT GGGTGTATAATC KPKEITPVNPGTSPLLRCCGTTTACTTTACGCGC AATGCAACCCAC AGGGCCGTGGTA YAAWTGGLAAVVLLCLVACCGATTCAGCGGATTT ACAAATGTCTGC TCGATAGCGGGG IFLICTA* ATGGAGTCCGGTACACCACAGGAGGATCT AACGGTTAATGC GAGACTTGGAGCTTTTT TGGGGACGATAC AACCCACACAAAGCCGTCATTAACCTGTA GGGCATCCACGT TGTCTGCACAGG CGGGAGACGCAGCGCCCTATCCCTACGTT AGGATCTTGGGG GCCATCCAGCATATATG AAACGGCGATGA ACGATACGGGCATTTAAAACATACAACAT CAGACATAAAAT TCCACGTTATCC GCTTTCAAGACGTGGTGTGTAAATGTGGA CTACGTTAAACG GTGGATGTGGATTGCGC CCAACGTCAATA GCGATGACAGACGGAAAATACTAAAGAGG CGGTGACGTGTT ATAAAATTGTAA ATCAGTTGGCCGAAATCTAAAGGAGATCT ATGTGGACCAAC AGTTACCGTTTTCAAGG TAATCCAAAACC GTCAATACGGTGTAAGAAGGAAGCGGACC CCAAGGCCAAAG ACGTGTTTAAAG AACCGTGGATTGTTGTAACTCATTGAGGT GAGATCTTAATC AACACGAGCACACTGTT GTCAGTGGAAGA CAAAACCCCAAGTGATGAACTCGAATTAG AAATCACCCGTT GCCAAAGACTCA ACCCCCCCGAGATTGAATACTTTACGCGC TTGAGGTGTCAG CCGGGTGTCTTGAAAGT ACCGATTCAGCG TGGAAGAAAATCACTTCGGACAGAAAAAC GATTTATGGAGT ACCCGTTTACTT AATACTTGGGTGTGTACCCGGTACACCGA TACGCGCACCGA ATTTGGAACATGCGCGG GACTTGGAGCTT TTCAGCGGATTTCTCCGATGGTACGTCTA TTTGCCGTCATT ATGGAGTCCGGT CCTACGCCACGTTTTTGAACCTGTACGGG ACACCGAGACTT GTCACCTGGAAAGGGGA AGACGCAGCGCC GGAGCTTTTTGCTGAAAAAACAAGAAACC CGCCATCCAGCA CGTCATTAACCT CTACGCCCGCAGTAACTTATATGTTTAAA GTACGGGAGACG CCTCAACCAAGAGGGGC ACATACAACATG CAGCGCCCGCCATGAGTTTCATATGTGGA CTTTCAAGACGT TCCAGCATATAT ATTACCACTCGCATGTAGGTGGTGGATGT GTTTAAAACATA TTTTCAGTTGGTGATAC GGATTGCGCGGA CAACATGCTTTCGTTTAGCTTGGCAATGC AAATACTAAAGA AAGACGTGGTGG ATCTTCAGTATAAGATAGGATCAGTTGGC TGGATGTGGATT CATGAAGCGCCATTTGA CGAAATCAGTTA GCGCGGAAAATATTTGCTGTTAGAGTGGT CCGTTTTCAAGG CTAAAGAGGATC TGTATGTCCCCATCGATTAAGAAGGAAGC AGTTGGCCGAAA CCTACATGTCAACCAAT GGACCAACCGTG TCAGTTACCGTTGCGGTTATATTCTACGT GATTGTTGTAAA TTCAAGGTAAGA GTTTGTATCATCCCAACCACGAGCACACT AGGAAGCGGACC GCACCCCAATGCCTCTC GTTTGATGAACT AACCGTGGATTGTCATATGAATTCCGGTT CGAATTAGACCC TTGTAAACACGA GTACATTTACCTCGCCACCCCGAGATTGA GCACACTGTTTG CATTTAGCCCAGCGTGT ACCGGGTGTCTT ATGAACTCGAATTGCAAGCACAGTGTATC GAAAGTACTTCG TAGACCCCCCCG AAAATTGTGAACATGCAGACAGAAAAACA AGATTGAACCGG GATAACTACACCGCATA ATACTTGGGTGT GTGTCTTGAAAGTTGTCTGGGAATATCTC GTACATTTGGAA TACTTCGGACAG ATATGGAGCCTAGCTTTCATGCGCGGCTC AAAAACAATACT GGTCTAATCTTACACGA CGATGGTACGTC TGGGTGTGTACACGGGGGCACCACGTTAA TACCTACGCCAC TTTGGAACATGC AGTTTGTAGATACACCCGTTTTTGGTCAC GCGGCTCCGATG GAGAGTTTGTCGGGATT CTGGAAAGGGGA GTACGTCTACCTATACGTTTTTGTGGTGT TGAAAAAACAAG ACGCCACGTTTT ATTTTAACGGGCATGTTAAACCCTACGCC TGGTCACCTGGA GAAGCCGTAGCATACAC CGCAGTAACTCC AAGGGGATGAAATGTTGTATCCACAGTAG TCAACCAAGAGG AAACAAGAAACC ATCATTTTGTAAACGCAGGCTGAGTTTCA CTACGCCCGCAG ATTGAAGAGCGTGGATT TATGTGGAATTA TAACTCCTCAACTCCGCCAACGGCCGGTC CCACTCGCATGT CAAGAGGGGCTG AGCCACCGGCGACTACTATTTTCAGTTGG AGTTTCATATGT AAACCCAAGGAAATTAC TGATACGTTTAG GGAATTACCACTCCCCGTAAACCCCGGAA CTTGGCAATGCA CGCATGTATTTT CGTCACCACTTCTACGATCTTCAGTATAA CAGTTGGTGATA TATGCCGCATGGACCGG GATACATGAAGC CGTTTAGCTTGGAGGGCTTGCAGCAGTAG GCCATTTGATTT CAATGCATCTTC TACTTTTATGTCTCGTAGCTGTTAGAGTG AGTATAAGATAC ATATTTTTAATCTGTAC GTTGTATGTCCC ATGAAGCGCCATGGCTTGA CATCGATCCTAC TTGATTTGCTGT ATGTCAACCAAT TAGAGTGGTTGT GCGGTTATATTCATGTCCCCATCG TACGTGTTTGTA ATCCTACATGTC TCATCCCAACGC AACCAATGCGGTACCCCAATGCCT TATATTCTACGT CTCTCATATGAA GTTTGTATCATC TTCCGGTTGTACCCAACGCACCCC ATTTACCTCGCC AATGCCTCTCTC ACATTTAGCCCA ATATGAATTCCGGCGTGTTGCAAG GTTGTACATTTA CACAGTGTATCA CCTCGCCACATT AAATTGTGAACATAGCCCAGCGTG TGCAGATAACTA TTGCAAGCACAG CACCGCATATTG TGTATCAAAATTTCTGGGAATATC GTGAACATGCAG TCATATGGAGCC ATAACTACACCG TAGCTTTGGTCTCATATTGTCTGG AATCTTACACGA GAATATCTCATA CGGGGGCACCAC TGGAGCCTAGCTGTTAAAGTTTGT TTGGTCTAATCT AGATACACCCGA TACACGACGGGG GAGTTTGTCGGGGCACCACGTTAA ATTATACGTTTT AGTTTGTAGATA TGTGGTGTATTT CACCCGAGAGTTTAACGGGCATGT TGTCGGGATTAT TGAAGCCGTAGC ACGTTTTTGTGG ATACACTGTTGTTGTATTTTAACG ATCCACAGTAGA GGCATGTTGAAG TCATTTTGTAAA CCGTAGCATACACGCAATTGAAGA CTGTTGTATCCA GCGTGGATTTCC CAGTAGATCATT GCCAACGGCCGGTTGTAAACGCAA TCAGCCACCGGC TTGAAGAGCGTG GACTACTAAACC GATTTCCGCCAACAAGGAAATTAC CGGCCGGTCAGC CCCCGTAAACCC CACCGGCGACTA CGGAACGTCACCCTAAACCCAAGG ACTTCTACGATA AAATTACCCCCG TGCCGCATGGAC TAAACCCCGGAACGGAGGGCTTGC CGTCACCACTTC AGCAGTAGTACT TACGATATGCCG TTTATGTCTCGTCATGGACCGGAG AATATTTTTAAT GGCTTGCAGCAG CTGTACGGCTTG TAGTACTTTTATATGATAATAGGC GTCTCGTAATAT TGGAGCCTCGGT TTTTAATCTGTA GGCCATGCTTCTCGGCTTGATGAT TGCCCCTTGGGC AATAGGCTGGAG CTCCCCCCAGCC CCTCGGTGGCCACCTCCTCCCCTT TGCTTCTTGCCC CCTGCACCCGTA CTTGGGCCTCCC CCCCCGTGGTCTCCCAGCCCCTCC TTGAATAAAGTC TCCCCTTCCTGC TGAGTGGGCGGC ACCCGTACCCCCAAAAAAAAAAAA GTGGTCTTTGAA AAAAAAAAAAAA TAAAGTCTGAGT AAAAAAAAAAAA GGGCGGCAAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAAAAAATCTAG SEQ ID NO: 25 SEQ ID NO: 26 SEQ ID NO: 27 SEQ ID NO: 28VZV-GE- TCAAGCTTTTGG MGTVNKPVVGVLMGFGI ATGGGGACAGTTAATAA G*GGGAAATAAGfull_with_ ACCCTCGTACAG ITGTLRITNPVRASVLR ACCTGTGGTGGGGGTAT AGAGAAAAGAAGAEAADA AAGCTAATACGA YDDFHIDEDKLDTNSVY TGATGGGGTTCGGAATT AGTAAGAAGAAA(SEQ CTCACTATAGGG EPYYHSDHAESSWVNRG ATCACGGGAACGTTGCG TATAAGAGCCACID NO: AAATAAGAGAGA ESSRKAYDHNSPYIWPR TATAACGAATCCGGTCA CATGGGGACAGT 58)AAAGAAGAGTAA NDYDGFLENAHEHHGVY GAGCATCCGTCTTGCGA TAATAAACCTGTGAAGAAATATAA NQGRGIDSGERLMQPTQ TACGATGATTTTCACAT GGTGGGGGTATTGAGCCACCATGG MSAQEDLGDDTGIHVIP CGATGAAGACAAACTGG GATGGGGTTCGGGGACAGTTAATA TLNGDDRHKIVNVDQRQ ATACAAACTCCGTATAT AATTATCACGGGAACCTGTGGTGG YGDVFKGDLNPKPQGQR GAGCCTTACTACCATTC AACGTTGCGTATGGGTATTGATGG LIEVSVEENHPFTLRAP AGATCATGCGGAGTCTT AACGAATCCGGTGGTTCGGAATTA IQRIYGVRYTETWSFLP CATGGGTAAATCGGGGA CAGAGCATCCGTTCACGGGAACGT SLTCTGDAAPAIQHICL GAGTCTTCGCGAAAAGC CTTGCGATACGATGCGTATAACGA KHTTCFQDVVVDVDCAE GTACGATCATAACTCAC TGATTTTCACATATCCGGTCAGAG NTKEDQLAEISYRFQGK CTTATATATGGCCACGT CGATGAAGACAACATCCGTCTTGC KEADQPWIVVNTSTLFD AATGATTATGATGGATT ACTGGATACAAAGATACGATGATT ELELDPPEIEPGVLKVL TTTAGAGAACGCACACG CTCCGTATATGATTCACATCGATG RTEKQYLGVYIWNMRGS AACACCATGGGGTGTAT GCCTTACTACCAAAGACAAACTGG DGTSTYATFLVTWKGDE AATCAGGGCCGTGGTAT TTCAGATCATGCATACAAACTCCG KTRNPTPAVTPQPRGAE CGATAGCGGGGAACGGT GGAGTCTTCATGTATATGAGCCTT FHMWNYHSHVFSVGDTF TAATGCAACCCACACAA GGTAAATCGGGGACTACCATTCAG SLAMHLQYKIHEAPFDL ATGTCTGCACAGGAGGA AGAGTCTTCGCGATCATGCGGAGT LLEWLYVPIDPTCQPMR TCTTGGGGACGATACGG AAAAGCGTACGACTTCATGGGTAA LYSTCLYHPNAPQCLSH GCATCCACGTTATCCCT TCATAACTCACCATCGGGGAGAGT MNSGCTFTSPHLAQRVA ACGTTAAACGGCGATGA TTATATATGGCCCTTCGCGAAAAG STVYQNCEHADNYTAYC CAGACATAAAATTGTAA ACGTAATGATTACGTACGATCATA LGISHMEPSFGLILHDG ATGTGGACCAACGTCAA TGATGGATTTTTACTCACCTTATA GTTLKFVDTPESLSGLY TACGGTGACGTGTTTAA AGAGAACGCACATATGGCCACGTA VFVVYFNGHVEAVAYTV AGGAGATCTTAATCCAA CGAACACCATGGATGATTATGATG VSTVDHFVNAIEERGFP AACCCCAAGGCCAAAGA GGTGTATAATCAGATTTTTAGAGA PTAGQPPATTKPKEITP CTCATTGAGGTGTCAGT GGGCCGTGGTATACGCACACGAAC VNPGTSPLLRYAAWTGG GGAAGAAAATCACCCGT CGATAGCGGGGAACCATGGGGTGT LAAVVLLCLVIFLICTA TTACTTTACGCGCACCG ACGGTTAATGCAATAATCAGGGCC KRMRVKAYRVDKSPYNQ ATTCAGCGGATTTATGG ACCCACACAAATGTGGTATCGATA SMYYAGLPVDDFEDAEA AGTCCGGTACACCGAGA GTCTGCACAGGAGCGGGGAACGGT ADAEEEFGNAIGGSHGG CTTGGAGCTTTTTGCCG GGATCTTGGGGATAATGCAACCCA SSYTVYIDKTR* TCATTAACCTGTACGGG CGATACGGGCAT CACAAATGTCTGAGACGCAGCGCCCGCCA CCACGTTATCCC CACAGGAGGATC TCCAGCATATATGTTTATACGTTAAACGG TTGGGGACGATA AAACATACAACATGCTT CGATGACAGACA CGGGCATCCACGTCAAGACGTGGTGGTGG TAAAATTGTAAA TTATCCCTACGT ATGTGGATTGCGCGGAATGTGGACCAACG TAAACGGCGATG AATACTAAAGAGGATCA TCAATACGGTGA ACAGACATAAAAGTTGGCCGAAATCAGTT CGTGTTTAAAGG TTGTAAATGTGG ACCGTTTTCAAGGTAAGAGATCTTAATCC ACCAACGTCAAT AAGGAAGCGGACCAACC AAAACCCCAAGG ACGGTGACGTGTGTGGATTGTTGTAAACA CCAAAGACTCAT TTAAAGGAGATC CGAGCACACTGTTTGATTGAGGTGTCAGT TTAATCCAAAAC GAACTCGAATTAGACCC GGAAGAAAATCA CCCAAGGCCAAACCCCGAGATTGAACCGG CCCGTTTACTTT GACTCATTGAGG GTGTCTTGAAAGTACTTACGCGCACCGAT TGTCAGTGGAAG CGGACAGAAAAACAATA TCAGCGGATTTA AAAATCACCCGTCTTGGGTGTGTACATTT TGGAGTCCGGTA TTACTTTACGCG GGAACATGCGCGGCTCCCACCGAGACTTG CACCGATTCAGC GATGGTACGTCTACCTA GAGCTTTTTGCC GGATTTATGGAGCGCCACGTTTTTGGTCA GTCATTAACCTG TCCGGTACACCG CCTGGAAAGGGGATGAATACGGGAGACGC AGACTTGGAGCT AAAACAAGAAACCCTAC AGCGCCCGCCAT TTTTGCCGTCATGCCCGCAGTAACTCCTC CCAGCATATATG TAACCTGTACGG AACCAAGAGGGGCTGAGTTTAAAACATAC GAGACGCAGCGC TTTCATATGTGGAATTA AACATGCTTTCA CCGCCATCCAGCCCACTCGCATGTATTTT AGACGTGGTGGT ATATATGTTTAA CAGTTGGTGATACGTTTGGATGTGGATTG AACATACAACAT AGCTTGGCAATGCATCT CGCGGAAAATAC GCTTTCAAGACGTCAGTATAAGATACATG TAAAGAGGATCA TGGTGGTGGATG AAGCGCCATTTGATTTGGTTGGCCGAAAT TGGATTGCGCGG CTGTTAGAGTGGTTGTA CAGTTACCGTTT AAAATACTAAAGTGTCCCCATCGATCCTA TCAAGGTAAGAA AGGATCAGTTGG CATGTCAACCAATGCGGGGAAGCGGACCA CCGAAATCAGTT TTATATTCTACGTGTTT ACCGTGGATTGT ACCGTTTTCAAGGTATCATCCCAACGCAC TGTAAACACGAG GTAAGAAGGAAG CCCAATGCCTCTCTCATCACACTGTTTGA CGGACCAACCGT ATGAATTCCGGTTGTAC TGAACTCGAATT GGATTGTTGTAAATTTACCTCGCCACATT AGACCCCCCCGA ACACGAGCACAC TAGCCCAGCGTGTTGCAGATTGAACCGGG TGTTTGATGAAC AGCACAGTGTATCAAAA TGTCTTGAAAGT TCGAATTAGACCTTGTGAACATGCAGATA ACTTCGGACAGA CCCCCGAGATTG ACTACACCGCATATTGTAAAACAATACTT AACCGGGTGTCT CTGGGAATATCTCATAT GGGTGTGTACAT TGAAAGTACTTCGGAGCCTAGCTTTGGTC TTGGAACATGCG GGACAGAAAAAC TAATCTTACACGACGGGCGGCTCCGATGG AATACTTGGGTG GGCACCACGTTAAAGTT TACGTCTACCTA TGTACATTTGGATGTAGATACACCCGAGA CGCCACGTTTTT ACATGCGCGGCT GTTTGTCGGGATTATACGGTCACCTGGAA CCGATGGTACGT GTTTTTGTGGTGTATTT AGGGGATGAAAA CTACCTACGCCATAACGGGCATGTTGAAG AACAAGAAACCC CGTTTTTGGTCA CCGTAGCATACACTGTTTACGCCCGCAGT CCTGGAAAGGGG GTATCCACAGTAGATCA AACTCCTCAACC ATGAAAAAACAATTTTGTAAACGCAATTG AAGAGGGGCTGA GAAACCCTACGC AAGAGCGTGGATTTCCGGTTTCATATGTG CCGCAGTAACTC CCAACGGCCGGTCAGCC GAATTACCACTC CTCAACCAAGAGACCGGCGACTACTAAAC GCATGTATTTTC GGGCTGAGTTTC CCAAGGAAATTACCCCCAGTTGGTGATAC ATATGTGGAATT GTAAACCCCGGAACGTC GTTTAGCTTGGC ACCACTCGCATGACCACTTCTACGATATG AATGCATCTTCA TATTTTCAGTTG CCGCATGGACCGGAGGGGTATAAGATACA GTGATACGTTTA CTTGCAGCAGTAGTACT TGAAGCGCCATT GCTTGGCAATGCTTTATGTCTCGTAATAT TGATTTGCTGTT ATCTTCAGTATA TTTTAATCTGTACGGCTAGAGTGGTTGTA AGATACATGAAG AAACGAATGAGGGTTAA TGTCCCCATCGA CGCCATTTGATTAGCCTATAGGGTAGACA TCCTACATGTCA TGCTGTTAGAGT AGTCCCCGTATAACCAAACCAATGCGGTT GGTTGTATGTCC AGCATGTATTACGCTGG ATATTCTACGTG CCATCGATCCTACCTTCCAGTGGACGATT TTTGTATCATCC CATGTCAACCAA TCGAGGACGCCGAAGCCCAACGCACCCCA TGCGGTTATATT GCCGATGCCGAAGAAGA ATGCCTCTCTCA CTACGTGTTTGTGTTTGGTAACGCGATTG TATGAATTCCGG ATCATCCCAACG GAGGGAGTCACGGGGGTTTGTACATTTAC CACCCCAATGCC TCGAGTTACACGGTGTA CTCGCCACATTT TCTCTCATATGATATAGATAAGACCCGGT AGCCCAGCGTGT ATTCCGGTTGTA GA TGCAAGCACAGT CATTTACCTCGCGTATCAAAATTG CACATTTAGCCC TGAACATGCAGA AGCGTGTTGCAA TAACTACACCGCGCACAGTGTATC ATATTGTCTGGG AAAATTGTGAAC AATATCTCATAT ATGCAGATAACTGGAGCCTAGCTT ACACCGCATATT TGGTCTAATCTT GTCTGGGAATAT ACACGACGGGGGCTCATATGGAGC CACCACGTTAAA CTAGCTTTGGTC GTTTGTAGATAC TAATCTTACACGACCCGAGAGTTT ACGGGGGCACCA GTCGGGATTATA CGTTAAAGTTTG CGTTTTTGTGGTTAGATACACCCG GTATTTTAACGG AGAGTTTGTCGG GCATGTTGAAGC GATTATACGTTTCGTAGCATACAC TTGTGGTGTATT TGTTGTATCCAC TTAACGGGCATG AGTAGATCATTTTTGAAGCCGTAG TGTAAACGCAAT CATACACTGTTG TGAAGAGCGTGG TATCCACAGTAGATTTCCGCCAAC ATCATTTTGTAA GGCCGGTCAGCC ACGCAATTGAAG ACCGGCGACTACAGCGTGGATTTC TAAACCCAAGGA CGCCAACGGCCG AATTACCCCCGT GTCAGCCACCGGAAACCCCGGAAC CGACTACTAAAC GTCACCACTTCT CCAAGGAAATTA ACGATATGCCGCCCCCCGTAAACC ATGGACCGGAGG CCGGAACGTCAC GCTTGCAGCAGT CACTTCTACGATAGTACTTTTATG ATGCCGCATGGA TCTCGTAATATT CCGGAGGGCTTG TTTAATCTGTACCAGCAGTAGTAC GGCTAAACGAAT TTTTATGTCTCG GAGGGTTAAAGC TAATATTTTTAACTATAGGGTAGA TCTGTACGGCTA CAAGTCCCCGTA AACGAATGAGGG TAACCAAAGCATTTAAAGCCTATA GTATTACGCTGG GGGTAGACAAGT CCTTCCAGTGGA CCCCGTATAACCCGATTTCGAGGA AAAGCATGTATT CGCCGAAGCCGC ACGCTGGCCTTC CGATGCCGAAGACAGTGGACGATT AGAGTTTGGTAA TCGAGGACGCCG CGCGATTGGAGG AAGCCGCCGATGGAGTCACGGGGG CCGAAGAAGAGT TTCGAGTTACAC TTGGTAACGCGA GGTGTATATAGATTGGAGGGAGTC TAAGACCCGGTG ACGGGGGTTCGA ATGATAATAGGC GTTACACGGTGTTGGAGCCTCGGT ATATAGATAAGA GGCCATGCTTCT CCCGGTGATGAT TGCCCCTTGGGCAATAGGCTGGAG CTCCCCCCAGCC CCTCGGTGGCCA CCTCCTCCCCTT TGCTTCTTGCCCCCTGCACCCGTA CTTGGGCCTCCC CCCCCGTGGTCT CCCAGCCCCTCC TTGAATAAAGTCTCCCCTTCCTGC TGAGTGGGCGGC ACCCGTACCCCC AAAAAAAAAAAA GTGGTCTTTGAAAAAAAAAAAAAA TAAAGTCTGAGT AAAAAAAAAAAA GGGCGGC AAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAATCTAG SEQ ID NO: 29SEQ ID NO: 30 SEQ ID NO: 31 SEQ ID NO: 32 VZV-GE- TCAAGCTTTTGGMGTVNKPVVGVLMGFGI ATGGGGACAGTTAATAA G*GGGAAATAAG full_with_ ACCCTCGTACAGITGTLRITNPVRASVLR ACCTGTGGTGGGGGTAT AGAGAAAAGAAG AEAADA AAGCTAATACGAYDDFHIDEDKLDTNSVY TGATGGGGTTCGGAATT AGTAAGAAGAAA (SEQ CTCACTATAGGGEPYYHSDHAESSWVNRG ATCACGGGAACGTTGCG TATAAGAGCCAC ID NO: AAATAAGAGAGAESSRKAYDHNSPYIWPR TATAACGAATCCGGTCA CATGGGGACAGT 58)_and_ AAAGAAGAGTAANDYDGFLENAHEHHGVY GAGCATCCGTCTTGCGA TAATAAACCTGT Y582G GAAGAAATATAANQGRGIDSGERLMQPTQ TACGATGATTTTCACAT GGTGGGGGTATT GAGCCACCATGGMSAQEDLGDDTGIHVIP CGATGAAGACAAACTGG GATGGGGTTCGG GGACAGTTAATATLNGDDRHKIVNVDQRQ ATACAAACTCCGTATAT AATTATCACGGG AACCTGTGGTGGYGDVFKGDLNPKPQGQR GAGCCTTACTACCATTC AACGTTGCGTAT GGGTATTGATGGLIEVSVEENHPFTLRAP AGATCATGCGGAGTCTT AACGAATCCGGT GGTTCGGAATTAIQRIYGVRYTETWSFLP CATGGGTAAATCGGGGA CAGAGCATCCGT TCACGGGAACGTSLTCTGDAAPAIQHICL GAGTCTTCGCGAAAAGC CTTGCGATACGA TGCGTATAACGAKHTTCFQDVVVDVDCAE GTACGATCATAACTCAC TGATTTTCACAT ATCCGGTCAGAGNTKEDQLAEISYRFQGK CTTATATATGGCCACGT CGATGAAGACAA CATCCGTCTTGCKEADQPWIVVNTSTLFD AATGATTATGATGGATT ACTGGATACAAA GATACGATGATTELELDPPEIEPGVLKVL TTTAGAGAACGCACACG CTCCGTATATGA TTCACATCGATGRTEKQYLGVYIWNMRGS AACACCATGGGGTGTAT GCCTTACTACCA AAGACAAACTGGDGTSTYATFLVTWKGDE AATCAGGGCCGTGGTAT TTCAGATCATGC ATACAAACTCCGKTRNPTPAVTPQPRGAE CGATAGCGGGGAACGGT GGAGTCTTCATG TATATGAGCCTTFHMWNYHSHVFSVGDTF TAATGCAACCCACACAA GGTAAATCGGGG ACTACCATTCAGSLAMHLQYKIHEAPFDL ATGTCTGCACAGGAGGA AGAGTCTTCGCG ATCATGCGGAGTLLEWLYVPIDPTCQPMR TCTTGGGGACGATACGG AAAAGCGTACGA CTTCATGGGTAALYSTCLYHPNAPQCLSH GCATCCACGTTATCCCT TCATAACTCACC ATCGGGGAGAGTMNSGCTFTSPHLAQRVA ACGTTAAACGGCGATGA TTATATATGGCC CTTCGCGAAAAGSTVYQNCEHADNYTAYC CAGACATAAAATTGTAA ACGTAATGATTA CGTACGATCATALGISHMEPSFGLILHDG ATGTGGACCAACGTCAA TGATGGATTTTT ACTCACCTTATAGTTLKFVDTPESLSGLY TACGGTGACGTGTTTAA AGAGAACGCACA TATGGCCACGTAVFVVYFNGHVEAVAYTV AGGAGATCTTAATCCAA CGAACACCATGG ATGATTATGATGVSTVDHFVNAIEERGFP AACCCCAAGGCCAAAGA GGTGTATAATCA GATTTTTAGAGAPTAGQPPATTKPKEITP CTCATTGAGGTGTCAGT GGGCCGTGGTAT ACGCACACGAACVNPGTSPLLRYAAWTGG GGAAGAAAATCACCCGT CGATAGCGGGGA ACCATGGGGTGTLAAVVLLCLVIFLICTA TTACTTTACGCGCACCG ACGGTTAATGCA ATAATCAGGGCCKRMRVKAYRVDKSPYNQ ATTCAGCGGATTTATGG ACCCACACAAAT GTGGTATCGATASMYGAGLPVDDFEDAEA AGTCCGGTACACCGAGA GTCTGCACAGGA GCGGGGAACGGTADAEEEFGNAIGGSHGG CTTGGAGCTTTTTGCCG GGATCTTGGGGA TAATGCAACCCASSYTVYIDKTR* TCATTAACCTGTACGGG CGATACGGGCAT CACAAATGTCTGAGACGCAGCGCCCGCCA CCACGTTATCCC CACAGGAGGATC TCCAGCATATATGTTTATACGTTAAACGG TTGGGGACGATA AAACATACAACATGCTT CGATGACAGACA CGGGCATCCACGTCAAGACGTGGTGGTGG TAAAATTGTAAA TTATCCCTACGT ATGTGGATTGCGCGGAATGTGGACCAACG TAAACGGCGATG AATACTAAAGAGGATCA TCAATACGGTGA ACAGACATAAAAGTTGGCCGAAATCAGTT CGTGTTTAAAGG TTGTAAATGTGG ACCGTTTTCAAGGTAAGAGATCTTAATCC ACCAACGTCAAT AAGGAAGCGGACCAACC AAAACCCCAAGG ACGGTGACGTGTGTGGATTGTTGTAAACA CCAAAGACTCAT TTAAAGGAGATC CGAGCACACTGTTTGATTGAGGTGTCAGT TTAATCCAAAAC GAACTCGAATTAGACCC GGAAGAAAATCA CCCAAGGCCAAACCCCGAGATTGAACCGG CCCGTTTACTTT GACTCATTGAGG GTGTCTTGAAAGTACTTACGCGCACCGAT TGTCAGTGGAAG CGGACAGAAAAACAATA TCAGCGGATTTA AAAATCACCCGTCTTGGGTGTGTACATTT TGGAGTCCGGTA TTACTTTACGCG GGAACATGCGCGGCTCCCACCGAGACTTG CACCGATTCAGC GATGGTACGTCTACCTA GAGCTTTTTGCC GGATTTATGGAGCGCCACGTTTTTGGTCA GTCATTAACCTG TCCGGTACACCG CCTGGAAAGGGGATGAATACGGGAGACGC AGACTTGGAGCT AAAACAAGAAACCCTAC AGCGCCCGCCAT TTTTGCCGTCATGCCCGCAGTAACTCCTC CCAGCATATATG TAACCTGTACGG AACCAAGAGGGGCTGAGTTTAAAACATAC GAGACGCAGCGC TTTCATATGTGGAATTA AACATGCTTTCA CCGCCATCCAGCCCACTCGCATGTATTTT AGACGTGGTGGT ATATATGTTTAA CAGTTGGTGATACGTTTGGATGTGGATTG AACATACAACAT AGCTTGGCAATGCATCT CGCGGAAAATAC GCTTTCAAGACGTCAGTATAAGATACATG TAAAGAGGATCA TGGTGGTGGATG AAGCGCCATTTGATTTGGTTGGCCGAAAT TGGATTGCGCGG CTGTTAGAGTGGTTGTA CAGTTACCGTTT AAAATACTAAAGTGTCCCCATCGATCCTA TCAAGGTAAGAA AGGATCAGTTGG CATGTCAACCAATGCGGGGAAGCGGACCA CCGAAATCAGTT TTATATTCTACGTGTTT ACCGTGGATTGT ACCGTTTTCAAGGTATCATCCCAACGCAC TGTAAACACGAG GTAAGAAGGAAG CCCAATGCCTCTCTCATCACACTGTTTGA CGGACCAACCGT ATGAATTCCGGTTGTAC TGAACTCGAATT GGATTGTTGTAAATTTACCTCGCCACATT AGACCCCCCCGA ACACGAGCACAC TAGCCCAGCGTGTTGCAGATTGAACCGGG TGTTTGATGAAC AGCACAGTGTATCAAAA TGTCTTGAAAGT TCGAATTAGACCTTGTGAACATGCAGATA ACTTCGGACAGA CCCCCGAGATTG ACTACACCGCATATTGTAAAACAATACTT AACCGGGTGTCT CTGGGAATATCTCATAT GGGTGTGTACAT TGAAAGTACTTCGGAGCCTAGCTTTGGTC TTGGAACATGCG GGACAGAAAAAC TAATCTTACACGACGGGCGGCTCCGATGG AATACTTGGGTG GGCACCACGTTAAAGTT TACGTCTACCTA TGTACATTTGGATGTAGATACACCCGAGA CGCCACGTTTTT ACATGCGCGGCT GTTTGTCGGGATTATACGGTCACCTGGAA CCGATGGTACGT GTTTTTGTGGTGTATTT AGGGGATGAAAA CTACCTACGCCATAACGGGCATGTTGAAG AACAAGAAACCC CGTTTTTGGTCA CCGTAGCATACACTGTTTACGCCCGCAGT CCTGGAAAGGGG GTATCCACAGTAGATCA AACTCCTCAACC ATGAAAAAACAATTTTGTAAACGCAATTG AAGAGGGGCTGA GAAACCCTACGC AAGAGCGTGGATTTCCGGTTTCATATGTG CCGCAGTAACTC CCAACGGCCGGTCAGCC GAATTACCACTC CTCAACCAAGAGACCGGCGACTACTAAAC GCATGTATTTTC GGGCTGAGTTTC CCAAGGAAATTACCCCCAGTTGGTGATAC ATATGTGGAATT GTAAACCCCGGAACGTC GTTTAGCTTGGC ACCACTCGCATGACCACTTCTACGATATG AATGCATCTTCA TATTTTCAGTTG CCGCATGGACCGGAGGGGTATAAGATACA GTGATACGTTTA CTTGCAGCAGTAGTACT TGAAGCGCCATT GCTTGGCAATGCTTTATGTCTCGTAATAT TGATTTGCTGTT ATCTTCAGTATA TTTTAATCTGTACGGCTAGAGTGGTTGTA AGATACATGAAG AAACGAATGAGGGTTAA TGTCCCCATCGA CGCCATTTGATTAGCCTATAGGGTAGACA TCCTACATGTCA TGCTGTTAGAGT AGTCCCCGTATAACCAAACCAATGCGGTT GGTTGTATGTCC AGCATGTATGGCGCTGG ATATTCTACGTG CCATCGATCCTACCTTCCAGTGGACGATT TTTGTATCATCC CATGTCAACCAA TCGAGGACGCCGAAGCCCAACGCACCCCA TGCGGTTATATT GCCGATGCCGAAGAAGA ATGCCTCTCTCA CTACGTGTTTGTGTTTGGTAACGCGATTG TATGAATTCCGG ATCATCCCAACG GAGGGAGTCACGGGGGTTTGTACATTTAC CACCCCAATGCC TCGAGTTACACGGTGTA CTCGCCACATTT TCTCTCATATGATATAGATAAGACCCGGT AGCCCAGCGTGT ATTCCGGTTGTA GA TGCAAGCACAGT CATTTACCTCGCGTATCAAAATTG CACATTTAGCCC TGAACATGCAGA AGCGTGTTGCAA TAACTACACCGCGCACAGTGTATC ATATTGTCTGGG AAAATTGTGAAC AATATCTCATAT ATGCAGATAACTGGAGCCTAGCTT ACACCGCATATT TGGTCTAATCTT GTCTGGGAATAT ACACGACGGGGGCTCATATGGAGC CACCACGTTAAA CTAGCTTTGGTC GTTTGTAGATAC TAATCTTACACGACCCGAGAGTTT ACGGGGGCACCA GTCGGGATTATA CGTTAAAGTTTG CGTTTTTGTGGTTAGATACACCCG GTATTTTAACGG AGAGTTTGTCGG GCATGTTGAAGC GATTATACGTTTCGTAGCATACAC TTGTGGTGTATT TGTTGTATCCAC TTAACGGGCATG AGTAGATCATTTTTGAAGCCGTAG TGTAAACGCAAT CATACACTGTTG TGAAGAGCGTGG TATCCACAGTAGATTTCCGCCAAC ATCATTTTGTAA GGCCGGTCAGCC ACGCAATTGAAG ACCGGCGACTACAGCGTGGATTTC TAAACCCAAGGA CGCCAACGGCCG AATTACCCCCGT GTCAGCCACCGGAAACCCCGGAAC CGACTACTAAAC GTCACCACTTCT CCAAGGAAATTA ACGATATGCCGCCCCCCGTAAACC ATGGACCGGAGG CCGGAACGTCAC GCTTGCAGCAGT CACTTCTACGATAGTACTTTTATG ATGCCGCATGGA TCTCGTAATATT CCGGAGGGCTTG TTTAATCTGTACCAGCAGTAGTAC GGCTAAACGAAT TTTTATGTCTCG GAGGGTTAAAGC TAATATTTTTAACTATAGGGTAGA TCTGTACGGCTA CAAGTCCCCGTA AACGAATGAGGG TAACCAAAGCATTTAAAGCCTATA GTATGGCGCTGG GGGTAGACAAGT CCTTCCAGTGGA CCCCGTATAACCCGATTTCGAGGA AAAGCATGTATG CGCCGAAGCCGC GCGCTGGCCTTC CGATGCCGAAGACAGTGGACGATT AGAGTTTGGTAA TCGAGGACGCCG CGCGATTGGAGG AAGCCGCCGATGGAGTCACGGGGG CCGAAGAAGAGT TTCGAGTTACAC TTGGTAACGCGA GGTGTATATAGATTGGAGGGAGTC TAAGACCCGGTG ACGGGGGTTCGA ATGATAATAGGC GTTACACGGTGTTGGAGCCTCGGT ATATAGATAAGA GGCCATGCTTCT CCCGGTGATGAT TGCCCCTTGGGCAATAGGCTGGAG CTCCCCCCAGCC CCTCGGTGGCCA CCTCCTCCCCTT TGCTTCTTGCCCCCTGCACCCGTA CTTGGGCCTCCC CCCCCGTGGTCT CCCAGCCCCTCC TTGAATAAAGTCTCCCCTTCCTGC TGAGTGGGCGGC ACCCGTACCCCC AAAAAAAAAAAA GTGGTCTTTGAAAAAAAAAAAAAA TAAAGTCTGAGT AAAAAAAAAAAA GGGCGGC AAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAATCTAG SEQ ID NO: 33SEQ ID NO: 34 SEQ ID NO: 35 SEQ ID NO: 36 VZV-GE- TCAAGCTTTTGGMGTVNKPVVGVLMGFGI ATGGGGACAGTTAATAA G*GGGAAATAAG truncated- ACCCTCGTACAGITGTLRITNPVRASVLR ACCTGTGGTGGGGGTAT AGAGAAAAGAAG delete_ AAGCTAATACGAYDDFHIDEDKLDTNSVY TGATGGGGTTCGGAATT AGTAAGAAGAAA from_574 CTCACTATAGGGEPYYHSDHAESSWVNRG ATCACGGGAACGTTGCG TATAAGAGCCAC AAATAAGAGAGAESSRKAYDHNSPYIWPR TATAACGAATCCGGTCA CATGGGGACAGT AAAGAAGAGTAANDYDGFLENAHEHHGVY GAGCATCCGTCTTGCGA TAATAAACCTGT GAAGAAATATAANQGRGIDSGERLMQPTQ TACGATGATTTTCACAT GGTGGGGGTATT GAGCCACCATGGMSAQEDLGDDTGIHVIP CGATGAAGACAAACTGG GATGGGGTTCGG GGACAGTTAATATLNGDDRHKIVNVDQRQ ATACAAACTCCGTATAT AATTATCACGGG AACCTGTGGTGGYGDVFKGDLNPKPQGQR GAGCCTTACTACCATTC AACGTTGCGTAT GGGTATTGATGGLIEVSVEENHPFTLRAP AGATCATGCGGAGTCTT AACGAATCCGGT GGTTCGGAATTAIQRIYGVRYTETWSFLP CATGGGTAAATCGGGGA CAGAGCATCCGT TCACGGGAACGTSLTCTGDAAPAIQHICL GAGTCTTCGCGAAAAGC CTTGCGATACGA TGCGTATAACGAKHTTCFQDVVVDVDCAE GTACGATCATAACTCAC TGATTTTCACAT ATCCGGTCAGAGNTKEDQLAEISYRFQGK CTTATATATGGCCACGT CGATGAAGACAA CATCCGTCTTGCKEADQPWIVVNTSTLFD AATGATTATGATGGATT ACTGGATACAAA GATACGATGATTELELDPPEIEPGVLKVL TTTAGAGAACGCACACG CTCCGTATATGA TTCACATCGATGRTEKQYLGVYIWNMRGS AACACCATGGGGTGTAT GCCTTACTACCA AAGACAAACTGGDGTSTYATFLVTWKGDE AATCAGGGCCGTGGTAT TTCAGATCATGC ATACAAACTCCGKTRNPTPAVTPQPRGAE CGATAGCGGGGAACGGT GGAGTCTTCATG TATATGAGCCTTFHMWNYHSHVFSVGDTF TAATGCAACCCACACAA GGTAAATCGGGG ACTACCATTCAGSLAMHLQYKIHEAPFDL ATGTCTGCACAGGAGGA AGAGTCTTCGCG ATCATGCGGAGTLLEWLYVPIDPTCQPMR TCTTGGGGACGATACGG AAAAGCGTACGA CTTCATGGGTAALYSTCLYHPNAPQCLSH GCATCCACGTTATCCCT TCATAACTCACC ATCGGGGAGAGTMNSGCTFTSPHLAQRVA ACGTTAAACGGCGATGA TTATATATGGCC CTTCGCGAAAAGSTVYQNCEHADNYTAYC CAGACATAAAATTGTAA ACGTAATGATTA CGTACGATCATALGISHMEPSFGLILHDG ATGTGGACCAACGTCAA TGATGGATTTTT ACTCACCTTATAGTTLKFVDTPESLSGLY TACGGTGACGTGTTTAA AGAGAACGCACA TATGGCCACGTAVFVVYFNGHVEAVAYTV AGGAGATCTTAATCCAA CGAACACCATGG ATGATTATGATGVSTVDHFVNAIEERGFP AACCCCAAGGCCAAAGA GGTGTATAATCA GATTTTTAGAGAPTAGQPPATTKPKEITP CTCATTGAGGTGTCAGT GGGCCGTGGTAT ACGCACACGAACVNPGTSPLLRYAAWTGG GGAAGAAAATCACCCGT CGATAGCGGGGA ACCATGGGGTGTLAAVVLLCLVIFLICTA TTACTTTACGCGCACCG ACGGTTAATGCA ATAATCAGGGCCKRMRVKAYRVDK* ATTCAGCGGATTTATGG ACCCACACAAAT GTGGTATCGATAAGTCCGGTACACCGAGA GTCTGCACAGGA GCGGGGAACGGT CTTGGAGCTTTTTGCCGGGATCTTGGGGA TAATGCAACCCA TCATTAACCTGTACGGG CGATACGGGCAT CACAAATGTCTGAGACGCAGCGCCCGCCA CCACGTTATCCC CACAGGAGGATC TCCAGCATATATGTTTATACGTTAAACGG TTGGGGACGATA AAACATACAACATGCTT CGATGACAGACA CGGGCATCCACGTCAAGACGTGGTGGTGG TAAAATTGTAAA TTATCCCTACGT ATGTGGATTGCGCGGAATGTGGACCAACG TAAACGGCGATG AATACTAAAGAGGATCA TCAATACGGTGA ACAGACATAAAAGTTGGCCGAAATCAGTT CGTGTTTAAAGG TTGTAAATGTGG ACCGTTTTCAAGGTAAGAGATCTTAATCC ACCAACGTCAAT AAGGAAGCGGACCAACC AAAACCCCAAGG ACGGTGACGTGTGTGGATTGTTGTAAACA CCAAAGACTCAT TTAAAGGAGATC CGAGCACACTGTTTGATTGAGGTGTCAGT TTAATCCAAAAC GAACTCGAATTAGACCC GGAAGAAAATCA CCCAAGGCCAAACCCCGAGATTGAACCGG CCCGTTTACTTT GACTCATTGAGG GTGTCTTGAAAGTACTTACGCGCACCGAT TGTCAGTGGAAG CGGACAGAAAAACAATA TCAGCGGATTTA AAAATCACCCGTCTTGGGTGTGTACATTT TGGAGTCCGGTA TTACTTTACGCG GGAACATGCGCGGCTCCCACCGAGACTTG CACCGATTCAGC GATGGTACGTCTACCTA GAGCTTTTTGCC GGATTTATGGAGCGCCACGTTTTTGGTCA GTCATTAACCTG TCCGGTACACCG CCTGGAAAGGGGATGAATACGGGAGACGC AGACTTGGAGCT AAAACAAGAAACCCTAC AGCGCCCGCCAT TTTTGCCGTCATGCCCGCAGTAACTCCTC CCAGCATATATG TAACCTGTACGG AACCAAGAGGGGCTGAGTTTAAAACATAC GAGACGCAGCGC TTTCATATGTGGAATTA AACATGCTTTCA CCGCCATCCAGCCCACTCGCATGTATTTT AGACGTGGTGGT ATATATGTTTAA CAGTTGGTGATACGTTTGGATGTGGATTG AACATACAACAT AGCTTGGCAATGCATCT CGCGGAAAATAC GCTTTCAAGACGTCAGTATAAGATACATG TAAAGAGGATCA TGGTGGTGGATG AAGCGCCATTTGATTTGGTTGGCCGAAAT TGGATTGCGCGG CTGTTAGAGTGGTTGTA CAGTTACCGTTT AAAATACTAAAGTGTCCCCATCGATCCTA TCAAGGTAAGAA AGGATCAGTTGG CATGTCAACCAATGCGGGGAAGCGGACCA CCGAAATCAGTT TTATATTCTACGTGTTT ACCGTGGATTGT ACCGTTTTCAAGGTATCATCCCAACGCAC TGTAAACACGAG GTAAGAAGGAAG CCCAATGCCTCTCTCATCACACTGTTTGA CGGACCAACCGT ATGAATTCCGGTTGTAC TGAACTCGAATT GGATTGTTGTAAATTTACCTCGCCACATT AGACCCCCCCGA ACACGAGCACAC TAGCCCAGCGTGTTGCAGATTGAACCGGG TGTTTGATGAAC AGCACAGTGTATCAAAA TGTCTTGAAAGT TCGAATTAGACCTTGTGAACATGCAGATA ACTTCGGACAGA CCCCCGAGATTG ACTACACCGCATATTGTAAAACAATACTT AACCGGGTGTCT CTGGGAATATCTCATAT GGGTGTGTACAT TGAAAGTACTTCGGAGCCTAGCTTTGGTC TTGGAACATGCG GGACAGAAAAAC TAATCTTACACGACGGGCGGCTCCGATGG AATACTTGGGTG GGCACCACGTTAAAGTT TACGTCTACCTA TGTACATTTGGATGTAGATACACCCGAGA CGCCACGTTTTT ACATGCGCGGCT GTTTGTCGGGATTATACGGTCACCTGGAA CCGATGGTACGT GTTTTTGTGGTGTATTT AGGGGATGAAAA CTACCTACGCCATAACGGGCATGTTGAAG AACAAGAAACCC CGTTTTTGGTCA CCGTAGCATACACTGTTTACGCCCGCAGT CCTGGAAAGGGG GTATCCACAGTAGATCA AACTCCTCAACC ATGAAAAAACAATTTTGTAAACGCAATTG AAGAGGGGCTGA GAAACCCTACGC AAGAGCGTGGATTTCCGGTTTCATATGTG CCGCAGTAACTC CCAACGGCCGGTCAGCC GAATTACCACTC CTCAACCAAGAGACCGGCGACTACTAAAC GCATGTATTTTC GGGCTGAGTTTC CCAAGGAAATTACCCCCAGTTGGTGATAC ATATGTGGAATT GTAAACCCCGGAACGTC GTTTAGCTTGGC ACCACTCGCATGACCACTTCTACGATATG AATGCATCTTCA TATTTTCAGTTG CCGCATGGACCGGAGGGGTATAAGATACA GTGATACGTTTA CTTGCAGCAGTAGTACT TGAAGCGCCATT GCTTGGCAATGCTTTATGTCTCGTAATAT TGATTTGCTGTT ATCTTCAGTATA TTTTAATCTGTACGGCTAGAGTGGTTGTA AGATACATGAAG AAACGAATGAGGGTTAA TGTCCCCATCGA CGCCATTTGATTAGCCTATAGGGTAGACA TCCTACATGTCA TGCTGTTAGAGT AGTGA ACCAATGCGGTTGGTTGTATGTCC ATATTCTACGTG CCATCGATCCTA TTTGTATCATCC CATGTCAACCAACAACGCACCCCA TGCGGTTATATT ATGCCTCTCTCA CTACGTGTTTGT TATGAATTCCGGATCATCCCAACG TTGTACATTTAC CACCCCAATGCC CTCGCCACATTT TCTCTCATATGAAGCCCAGCGTGT ATTCCGGTTGTA TGCAAGCACAGT CATTTACCTCGC GTATCAAAATTGCACATTTAGCCC TGAACATGCAGA AGCGTGTTGCAA TAACTACACCGC GCACAGTGTATCATATTGTCTGGG AAAATTGTGAAC AATATCTCATAT ATGCAGATAACT GGAGCCTAGCTTACACCGCATATT TGGTCTAATCTT GTCTGGGAATAT ACACGACGGGGG CTCATATGGAGCCACCACGTTAAA CTAGCTTTGGTC GTTTGTAGATAC TAATCTTACACG ACCCGAGAGTTTACGGGGGCACCA GTCGGGATTATA CGTTAAAGTTTG CGTTTTTGTGGT TAGATACACCCGGTATTTTAACGG AGAGTTTGTCGG GCATGTTGAAGC GATTATACGTTT CGTAGCATACACTTGTGGTGTATT TGTTGTATCCAC TTAACGGGCATG AGTAGATCATTT TTGAAGCCGTAGTGTAAACGCAAT CATACACTGTTG TGAAGAGCGTGG TATCCACAGTAG ATTTCCGCCAACATCATTTTGTAA GGCCGGTCAGCC ACGCAATTGAAG ACCGGCGACTAC AGCGTGGATTTCTAAACCCAAGGA CGCCAACGGCCG AATTACCCCCGT GTCAGCCACCGG AAACCCCGGAACCGACTACTAAAC GTCACCACTTCT CCAAGGAAATTA ACGATATGCCGC CCCCCGTAAACCATGGACCGGAGG CCGGAACGTCAC GCTTGCAGCAGT CACTTCTACGAT AGTACTTTTATGATGCCGCATGGA TCTCGTAATATT CCGGAGGGCTTG TTTAATCTGTAC CAGCAGTAGTACGGCTAAACGAAT TTTTATGTCTCG GAGGGTTAAAGC TAATATTTTTAA CTATAGGGTAGATCTGTACGGCTA CAAGTGATGATA AACGAATGAGGG ATAGGCTGGAGC TTAAAGCCTATACTCGGTGGCCAT GGGTAGACAAGT GCTTCTTGCCCC GATGATAATAGG TTGGGCCTCCCCCTGGAGCCTCGG CCAGCCCCTCCT TGGCCATGCTTC CCCCTTCCTGCA TTGCCCCTTGGGCCCGTACCCCCG CCTCCCCCCAGC TGGTCTTTGAAT CCCTCCTCCCCT AAAGTCTGAGTGTCCTGCACCCGT GGCGGCAAAAAA ACCCCCGTGGTC AAAAAAAAAAAA TTTGAATAAAGTAAAAAAAAAAAA CTGAGTGGGCGG AAAAAAAAAAAA C AAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAATC TAG SEQ ID NO: 37 SEQ ID NO: 38SEQ ID NO: 39 SEQ ID NO: 40 VZV-GE- TCAAGCTTTTGG MGTVNKPVVGVLMGFGIATGGGGACAGTTAATAA G*GGGAAATAAG truncated- ACCCTCGTACAG ITGTLRITNPVRASVLRACCTGTGGTGGGGGTAT AGAGAAAAGAAG delete_ AAGCTAATACGA YDDFHIDEDKLDTNSVYTGATGGGGTTCGGAATT AGTAAGAAGAAA from_574_-_ CTCACTATAGGGEPYYHSDHAESSWVNRG ATCACGGGAACGTTGCG TATAAGAGCCAC Y569A AAATAAGAGAGAESSRKAYDHNSPYIWPR TATAACGAATCCGGTCA CATGGGGACAGT AAAGAAGAGTAANDYDGFLENAHEHHGVY GAGCATCCGTCTTGCGA TAATAAACCTGT GAAGAAATATAANQGRGIDSGERLMQPTQ TACGATGATTTTCACAT GGTGGGGGTATT GAGCCACCATGGMSAQEDLGDDTGIHVIP CGATGAAGACAAACTGG GATGGGGTTCGG GGACAGTTAATATLNGDDRHKIVNVDQRQ ATACAAACTCCGTATAT AATTATCACGGG AACCTGTGGTGGYGDVFKGDLNPKPQGQR GAGCCTTACTACCATTC AACGTTGCGTAT GGGTATTGATGGLIEVSVEENHPFTLRAP AGATCATGCGGAGTCTT AACGAATCCGGT GGTTCGGAATTAIQRIYGVRYTETWSFLP CATGGGTAAATCGGGGA CAGAGCATCCGT TCACGGGAACGTSLTCTGDAAPAIQHICL GAGTCTTCGCGAAAAGC CTTGCGATACGA TGCGTATAACGAKHTTCFQDVVVDVDCAE GTACGATCATAACTCAC TGATTTTCACAT ATCCGGTCAGAGNTKEDQLAEISYRFQGK CTTATATATGGCCACGT CGATGAAGACAA CATCCGTCTTGCKEADQPWIVVNTSTLFD AATGATTATGATGGATT ACTGGATACAAA GATACGATGATTELELDPPEIEPGVLKVL TTTAGAGAACGCACACG CTCCGTATATGA TTCACATCGATGRTEKQYLGVYIWNMRGS AACACCATGGGGTGTAT GCCTTACTACCA AAGACAAACTGGDGTSTYATFLVTWKGDE AATCAGGGCCGTGGTAT TTCAGATCATGC ATACAAACTCCGKTRNPTPAVTPQPRGAE CGATAGCGGGGAACGGT GGAGTCTTCATG TATATGAGCCTTFHMWNYHSHVFSVGDTF TAATGCAACCCACACAA GGTAAATCGGGG ACTACCATTCAGSLAMHLQYKIHEAPFDL ATGTCTGCACAGGAGGA AGAGTCTTCGCG ATCATGCGGAGTLLEWLYVPIDPTCQPMR TCTTGGGGACGATACGG AAAAGCGTACGA CTTCATGGGTAALYSTCLYHPNAPQCLSH GCATCCACGTTATCCCT TCATAACTCACC ATCGGGGAGAGTMNSGCTFTSPHLAQRVA ACGTTAAACGGCGATGA TTATATATGGCC CTTCGCGAAAAGSTVYQNCEHADNYTAYC CAGACATAAAATTGTAA ACGTAATGATTA CGTACGATCATALGISHMEPSFGLILHDG ATGTGGACCAACGTCAA TGATGGATTTTT ACTCACCTTATAGTTLKFVDTPESLSGLY TACGGTGACGTGTTTAA AGAGAACGCACA TATGGCCACGTAVFVVYFNGHVEAVAYTV AGGAGATCTTAATCCAA CGAACACCATGG ATGATTATGATGVSTVDHFVNAIEERGFP AACCCCAAGGCCAAAGA GGTGTATAATCA GATTTTTAGAGAPTAGQPPATTKPKEITP CTCATTGAGGTGTCAGT GGGCCGTGGTAT ACGCACACGAACVNPGTSPLLRYAAWTGG GGAAGAAAATCACCCGT CGATAGCGGGGA ACCATGGGGTGTLAAVVLLCLVIFLICTA TTACTTTACGCGCACCG ACGGTTAATGCA ATAATCAGGGCCKRMRVKAARVDK* ATTCAGCGGATTTATGG ACCCACACAAAT GTGGTATCGATAAGTCCGGTACACCGAGA GTCTGCACAGGA GCGGGGAACGGT CTTGGAGCTTTTTGCCGGGATCTTGGGGA TAATGCAACCCA TCATTAACCTGTACGGG CGATACGGGCAT CACAAATGTCTGAGACGCAGCGCCCGCCA CCACGTTATCCC CACAGGAGGATC TCCAGCATATATGTTTATACGTTAAACGG TTGGGGACGATA AAACATACAACATGCTT CGATGACAGACA CGGGCATCCACGTCAAGACGTGGTGGTGG TAAAATTGTAAA TTATCCCTACGT ATGTGGATTGCGCGGAATGTGGACCAACG TAAACGGCGATG AATACTAAAGAGGATCA TCAATACGGTGA ACAGACATAAAAGTTGGCCGAAATCAGTT CGTGTTTAAAGG TTGTAAATGTGG ACCGTTTTCAAGGTAAGAGATCTTAATCC ACCAACGTCAAT AAGGAAGCGGACCAACC AAAACCCCAAGG ACGGTGACGTGTGTGGATTGTTGTAAACA CCAAAGACTCAT TTAAAGGAGATC CGAGCACACTGTTTGATTGAGGTGTCAGT TTAATCCAAAAC GAACTCGAATTAGACCC GGAAGAAAATCA CCCAAGGCCAAACCCCGAGATTGAACCGG CCCGTTTACTTT GACTCATTGAGG GTGTCTTGAAAGTACTTACGCGCACCGAT TGTCAGTGGAAG CGGACAGAAAAACAATA TCAGCGGATTTA AAAATCACCCGTCTTGGGTGTGTACATTT TGGAGTCCGGTA TTACTTTACGCG GGAACATGCGCGGCTCCCACCGAGACTTG CACCGATTCAGC GATGGTACGTCTACCTA GAGCTTTTTGCC GGATTTATGGAGCGCCACGTTTTTGGTCA GTCATTAACCTG TCCGGTACACCG CCTGGAAAGGGGATGAATACGGGAGACGC AGACTTGGAGCT AAAACAAGAAACCCTAC AGCGCCCGCCAT TTTTGCCGTCATGCCCGCAGTAACTCCTC CCAGCATATATG TAACCTGTACGG AACCAAGAGGGGCTGAGTTTAAAACATAC GAGACGCAGCGC TTTCATATGTGGAATTA AACATGCTTTCA CCGCCATCCAGCCCACTCGCATGTATTTT AGACGTGGTGGT ATATATGTTTAA CAGTTGGTGATACGTTTGGATGTGGATTG AACATACAACAT AGCTTGGCAATGCATCT CGCGGAAAATAC GCTTTCAAGACGTCAGTATAAGATACATG TAAAGAGGATCA TGGTGGTGGATG AAGCGCCATTTGATTTGGTTGGCCGAAAT TGGATTGCGCGG CTGTTAGAGTGGTTGTA CAGTTACCGTTT AAAATACTAAAGTGTCCCCATCGATCCTA TCAAGGTAAGAA AGGATCAGTTGG CATGTCAACCAATGCGGGGAAGCGGACCA CCGAAATCAGTT TTATATTCTACGTGTTT ACCGTGGATTGT ACCGTTTTCAAGGTATCATCCCAACGCAC TGTAAACACGAG GTAAGAAGGAAG CCCAATGCCTCTCTCATCACACTGTTTGA CGGACCAACCGT ATGAATTCCGGTTGTAC TGAACTCGAATT GGATTGTTGTAAATTTACCTCGCCACATT AGACCCCCCCGA ACACGAGCACAC TAGCCCAGCGTGTTGCAGATTGAACCGGG TGTTTGATGAAC AGCACAGTGTATCAAAA TGTCTTGAAAGT TCGAATTAGACCTTGTGAACATGCAGATA ACTTCGGACAGA CCCCCGAGATTG ACTACACCGCATATTGTAAAACAATACTT AACCGGGTGTCT CTGGGAATATCTCATAT GGGTGTGTACAT TGAAAGTACTTCGGAGCCTAGCTTTGGTC TTGGAACATGCG GGACAGAAAAAC TAATCTTACACGACGGGCGGCTCCGATGG AATACTTGGGTG GGCACCACGTTAAAGTT TACGTCTACCTA TGTACATTTGGATGTAGATACACCCGAGA CGCCACGTTTTT ACATGCGCGGCT GTTTGTCGGGATTATACGGTCACCTGGAA CCGATGGTACGT GTTTTTGTGGTGTATTT AGGGGATGAAAA CTACCTACGCCATAACGGGCATGTTGAAG AACAAGAAACCC CGTTTTTGGTCA CCGTAGCATACACTGTTTACGCCCGCAGT CCTGGAAAGGGG GTATCCACAGTAGATCA AACTCCTCAACC ATGAAAAAACAATTTTGTAAACGCAATTG AAGAGGGGCTGA GAAACCCTACGC AAGAGCGTGGATTTCCGGTTTCATATGTG CCGCAGTAACTC CCAACGGCCGGTCAGCC GAATTACCACTC CTCAACCAAGAGACCGGCGACTACTAAAC GCATGTATTTTC GGGCTGAGTTTC CCAAGGAAATTACCCCCAGTTGGTGATAC ATATGTGGAATT GTAAACCCCGGAACGTC GTTTAGCTTGGC ACCACTCGCATGACCACTTCTACGATATG AATGCATCTTCA TATTTTCAGTTG CCGCATGGACCGGAGGGGTATAAGATACA GTGATACGTTTA CTTGCAGCAGTAGTACT TGAAGCGCCATT GCTTGGCAATGCTTTATGTCTCGTAATAT TGATTTGCTGTT ATCTTCAGTATA TTTTAATCTGTACGGCTAGAGTGGTTGTA AGATACATGAAG AAACGAATGAGGGTTAA TGTCCCCATCGA CGCCATTTGATTAGCCGCCAGGGTAGACA TCCTACATGTCA TGCTGTTAGAGT AGTGA ACCAATGCGGTTGGTTGTATGTCC ATATTCTACGTG CCATCGATCCTA TTTGTATCATCC CATGTCAACCAACAACGCACCCCA TGCGGTTATATT ATGCCTCTCTCA CTACGTGTTTGT TATGAATTCCGGATCATCCCAACG TTGTACATTTAC CACCCCAATGCC CTCGCCACATTT TCTCTCATATGAAGCCCAGCGTGT ATTCCGGTTGTA TGCAAGCACAGT CATTTACCTCGC GTATCAAAATTGCACATTTAGCCC TGAACATGCAGA AGCGTGTTGCAA TAACTACACCGC GCACAGTGTATCATATTGTCTGGG AAAATTGTGAAC AATATCTCATAT ATGCAGATAACT GGAGCCTAGCTTACACCGCATATT TGGTCTAATCTT GTCTGGGAATAT ACACGACGGGGG CTCATATGGAGCCACCACGTTAAA CTAGCTTTGGTC GTTTGTAGATAC TAATCTTACACG ACCCGAGAGTTTACGGGGGCACCA GTCGGGATTATA CGTTAAAGTTTG CGTTTTTGTGGT TAGATACACCCGGTATTTTAACGG AGAGTTTGTCGG GCATGTTGAAGC GATTATACGTTT CGTAGCATACACTTGTGGTGTATT TGTTGTATCCAC TTAACGGGCATG AGTAGATCATTT TTGAAGCCGTAGTGTAAACGCAAT CATACACTGTTG TGAAGAGCGTGG TATCCACAGTAG ATTTCCGCCAACATCATTTTGTAA GGCCGGTCAGCC ACGCAATTGAAG ACCGGCGACTAC AGCGTGGATTTCTAAACCCAAGGA CGCCAACGGCCG AATTACCCCCGT GTCAGCCACCGG AAACCCCGGAACCGACTACTAAAC GTCACCACTTCT CCAAGGAAATTA ACGATATGCCGC CCCCCGTAAACCATGGACCGGAGG CCGGAACGTCAC GCTTGCAGCAGT CACTTCTACGAT AGTACTTTTATGATGCCGCATGGA TCTCGTAATATT CCGGAGGGCTTG TTTAATCTGTAC CAGCAGTAGTACGGCTAAACGAAT TTTTATGTCTCG GAGGGTTAAAGC TAATATTTTTAA CGCCAGGGTAGATCTGTACGGCTA CAAGTGATGATA AACGAATGAGGG ATAGGCTGGAGC TTAAAGCCGCCACTCGGTGGCCAT GGGTAGACAAGT GCTTCTTGCCCC GATGATAATAGG TTGGGCCTCCCCCTGGAGCCTCGG CCAGCCCCTCCT TGGCCATGCTTC CCCCTTCCTGCA TTGCCCCTTGGGCCCGTACCCCCG CCTCCCCCCAGC TGGTCTTTGAAT CCCTCCTCCCCT AAAGTCTGAGTGTCCTGCACCCGT GGCGGCAAAAAA ACCCCCGTGGTC AAAAAAAAAAAA TTTGAATAAAGTAAAAAAAAAAAA CTGAGTGGGCGG AAAAAAAAAAAA C AAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAATC TAG SEQ ID NO: 2 SEQ ID NO: 42SEQ ID NO: 43 SEQ ID NO: 44 VZV-GI- TCAAGCTTTTGG MFLIQCLISAVIFYIQVATGTTTTTAATCCAATG G*GGGAAATAAG full ACCCTCGTACAG TNALIFKGDHVSLQVNSTTTGATATCGGCCGTTA AGAGAAAAGAAG AAGCTAATACGA SLTSILIPMQNDNYTEITATTTTACATACAAGTG AGTAAGAAGAAA CTCACTATAGGG KGQLVFigEQLPTGTNYACCAACGCTTTGATCTT TATAAGAGCCAC AAATAAGAGAGA SGTLELLYADTVAFCFRCAAGGGCGACCACGTGA CATGTTTTTAAT AAAGAAGAGTAA SVQVIRYDGCPRIRTSAGCTTGCAAGTTAACAGC CCAATGTTTGAT GAAGAAATATAA FISCRYKHSWHYGNSTDAGTCTCACGTCTATCCT ATCGGCCGTTAT GAGCCACCATGT RISTEPDAGVMLKITKPTATTCCCATGCAAAATG ATTTTACATACA TTTTAATCCAAT GINDAGVYVLLVRLDHSATAATTATACAGAGATA AGTGACCAACGC GTTTGATATCGG RSTDGFILGVNVYTAGSAAAGGACAGCTTGTCTT TTTGATCTTCAA CCGTTATATTTT HHNIHGVIYTSPSLQNGTATTGGAGAGCAACTAC GGGCGACCACGT ACATACAAGTGA YSTRALFQQARLCDLPACTACCGGGACAAACTAT GAGCTTGCAAGT CCAACGCTTTGA TPKGSGTSLFQHMLDLRAGCGGAACACTGGAACT TAACAGCAGTCT TCTTCAAGGGCG AGKSLEDNPWLHEDVVTGTTATACGCGGATACGG CACGTCTATCCT ACCACGTGAGCT TETKSVVKEGIENHVYPTGGCGTTTTGTTTCCGG TATTCCCATGCA TGCAAGTTAACA TDMSTLPEKSLNDPPENTCAGTACAAGTAATAAG AAATGATAATTA GCAGTCTCACGT LLIIIPIVASVMILTAMATACGACGGATGTCCCC TACAGAGATAAA CTATCCTTATTC VIVIVISVKRRRIKKHPGGATTAGAACGAGCGCT AGGACAGCTTGT CCATGCAAAATG IYRPNTKTRRGIQNATPTTTATTTCGTGTAGGTA CTTTATTGGAGA ATAATTATACAG ESDVMLEAAIAQLATIRCAAACATTCGTGGCATT GCAACTACCTAC AGATAAAAGGAC EESPPHSVVNPFVK*ATGGTAACTCAACGGAT CGGGACAAACTA AGCTTGTCTTTA CGGATATCAACAGAGCCTAGCGGAACACT TTGGAGAGCAAC GGATGCTGGTGTAATGT GGAACTGTTATA TACCTACCGGGATGAAAATTACCAAACCG CGCGGATACGGT CAAACTATAGCG GGAATAAATGATGCTGGGGCGTTTTGTTT GAACACTGGAAC TGTGTATGTACTTCTTG CCGGTCAGTACA TGTTATACGCGGTTCGGTTAGACCATAGC AGTAATAAGATA ATACGGTGGCGT AGATCCACCGATGGTTTCGACGGATGTCC TTTGTTTCCGGT CATTCTTGGTGTAAATG CCGGATTAGAAC CAGTACAAGTAATATATACAGCGGGCTCG GAGCGCTTTTAT TAAGATACGACG CATCACAACATTCACGGTTCGTGTAGGTA GATGTCCCCGGA GGTTATCTACACTTCTC CAAACATTCGTG TTAGAACGAGCGCATCTCTACAGAATGGA GCATTATGGTAA CTTTTATTTCGT TATTCTACAAGAGCCCTCTCAACGGATCG GTAGGTACAAAC TTTTCAACAAGCTCGTT GATATCAACAGA ATTCGTGGCATTTGTGTGATTTACCCGCG GCCGGATGCTGG ATGGTAACTCAA ACACCCAAAGGGTCCGGTGTAATGTTGAA CGGATCGGATAT TACCTCCCTGTTTCAAC AATTACCAAACC CAACAGAGCCGGATATGCTTGATCTTCGT GGGAATAAATGA ATGCTGGTGTAA GCCGGTAAATCGTTAGATGCTGGTGTGTA TGTTGAAAATTA GGATAACCCTTGGTTAC TGTACTTCTTGT CCAAACCGGGAAATGAGGACGTTGTTACG TCGGTTAGACCA TAAATGATGCTG ACAGAAACTAAGTCCGTTAGCAGATCCAC GTGTGTATGTAC TGTTAAGGAGGGGATAG CGATGGTTTCAT TTCTTGTTCGGTAAAATCACGTATATCCA TCTTGGTGTAAA TAGACCATAGCA ACGGATATGTCCACGTTTGTATATACAGC GATCCACCGATG ACCCGAAAAGTCCCTTA GGGCTCGCATCA GTTTCATTCTTGATGATCCTCCAGAAAAT CAACATTCACGG GTGTAAATGTAT CTACTTATAATTATTCCGGTTATCTACAC ATACAGCGGGCT TATAGTAGCGTCTGTCA TTCTCCATCTCT CGCATCACAACATGATCCTCACCGCCATG ACAGAATGGATA TTCACGGGGTTA GTTATTGTTATTGTAATTTCTACAAGAGC TCTACACTTCTC AAGCGTTAAGCGACGTA CCTTTTTCAACA CATCTCTACAGAGAATTAAAAAACATCCA AGCTCGTTTGTG ATGGATATTCTA ATTTATCGCCCAAATACTGATTTACCCGC CAAGAGCCCTTT AAAAACAAGAAGGGGCA GACACCCAAAGG TTCAACAAGCTCTACAAAATGCGACACCA GTCCGGTACCTC GTTTGTGTGATT GAATCCGATGTGATGTTCCTGTTTCAACA TACCCGCGACAC GGAGGCCGCCATTGCAC TATGCTTGATCT CCAAAGGGTCCGAACTAGCAACGATTCGC TCGTGCCGGTAA GTACCTCCCTGT GAAGAATCCCCCCCACAATCGTTAGAGGA TTCAACATATGC TTCCGTTGTAAACCCGT TAACCCTTGGTT TTGATCTTCGTGTTGTTAAATAG ACATGAGGACGT CCGGTAAATCGT TGTTACGACAGA TAGAGGATAACCAACTAAGTCCGT CTTGGTTACATG TGTTAAGGAGGG AGGACGTTGTTA GATAGAAAATCACGACAGAAACTA CGTATATCCAAC AGTCCGTTGTTA GGATATGTCCAC AGGAGGGGATAGGTTACCCGAAAA AAAATCACGTAT GTCCCTTAATGA ATCCAACGGATA TCCTCCAGAAAATGTCCACGTTAC TCTACTTATAAT CCGAAAAGTCCC TATTCCTATAGT TTAATGATCCTCAGCGTCTGTCAT CAGAAAATCTAC GATCCTCACCGC TTATAATTATTC CATGGTTATTGTCTATAGTAGCGT TATTGTAATAAG CTGTCATGATCC CGTTAAGCGACG TCACCGCCATGGTAGAATTAAAAA TTATTGTTATTG ACATCCAATTTA TAATAAGCGTTA TCGCCCAAATACAGCGACGTAGAA AAAAACAAGAAG TTAAAAAACATC GGGCATACAAAA CAATTTATCGCCTGCGACACCAGA CAAATACAAAAA ATCCGATGTGAT CAAGAAGGGGCA GTTGGAGGCCGCTACAAAATGCGA CATTGCACAACT CACCAGAATCCG AGCAACGATTCG ATGTGATGTTGGCGAAGAATCCCC AGGCCGCCATTG CCCACATTCCGT CACAACTAGCAA TGTAAACCCGTTCGATTCGCGAAG TGTTAAATAGTG AATCCCCCCCAC ATAATAGGCTGG ATTCCGTTGTAAAGCCTCGGTGGC ACCCGTTTGTTA CATGCTTCTTGC AATAGTGATAAT CCCTTGGGCCTCAGGCTGGAGCCT CCCCCAGCCCCT CGGTGGCCATGC CCTCCCCTTCCT TTCTTGCCCCTTGCACCCGTACCC GGGCCTCCCCCC CCGTGGTCTTTG AGCCCCTCCTCC AATAAAGTCTGACCTTCCTGCACC GTGGGCGGCAAA CGTACCCCCGTG AAAAAAAAAAAA GTCTTTGAATAAAAAAAAAAAAAA AGTCTGAGTGGG AAAAAAAAAAAA CGGC AAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA ATCTAG SEQ ID NO: 60SEQ ID NO: 61 SEQ ID NO: 62 SEQ ID NO: 63 VZV-GE- GGGAAATAAGAGMGTVNKPVVGVLMGFGI ATGGGCACCGTGAACAA GGGAAATAAGAG truncated- AGAAAAGAAGAGITGTLRITNPVRASVLR GCCCGTCGTGGGCGTGC AGAAAAGAAGAG delete_ TAAGAAGAAATAYDDFHIDEDKLDTNSVY TGATGGGCTTCGGCATC TAAGAAGAAATA from_574_-_TAAGAGCCACCA EPYYHSDHAESSWVNRG ATCACCGGCACCCTGCG TAAGAGCCACCA Y569ATGGGCACCGTGA ESSRKAYDHNSPYIWPR GATCACCAATCCTGTGC TGGGCACCGTGA VariantACAAGCCCGTCG NDYDGFLENAHEHHGVY GGGCCAGCGTGCTGAGA ACAAGCCCGTCG 1TGGGCGTGCTGA NQGRGIDSGERLMQPTQ TACGACGACTTCCACAT TGGGCGTGCTGATGGGCTTCGGCA MSAQEDLGDDTGIHVIP CGACGAGGACAAGCTGG TGGGCTTCGGCATCATCACCGGCA TLNGDDRHKIVNVDQRQ ACACCAACAGCGTGTAC TCATCACCGGCACCCTGCGGATCA YGDVFKGDLNPKPQGQR GAGCCCTACTACCACAG CCCTGCGGATCACCAATCCTGTGC LIEVSVEENHPFTLRAP CGACCACGCCGAGAGCA CCAATCCTGTGCGGGCCAGCGTGC IQRIYGVRYTETWSFLP GCTGGGTCAACAGAGGC GGGCCAGCGTGCTGAGATACGACG SLTCTGDAAPAIQHICL GAGTCCAGCCGGAAGGC TGAGATACGACGACTTCCACATCG KHTTCFQDVVVDVDCAE CTACGACCACAACAGCC ACTTCCACATCGACGAGGACAAGC NTKEDQLAEISYRFQGK CCTACATCTGGCCCCGG ACGAGGACAAGCTGGACACCAACA KEADQPWIVVNTSTLFD AACGACTACGACGGCTT TGGACACCAACAGCGTGTACGAGC ELELDPPEIEPGVLKVL CCTGGAAAATGCCCACG GCGTGTACGAGCCCTACTACCACA RTEKQYLGVYIWNMRGS AGCACCACGGCGTGTAC CCTACTACCACAGCGACCACGCCG DGTSTYATFLVTWKGDE AACCAGGGCAGAGGCAT GCGACCACGCCGAGAGCAGCTGGG KTRNPTPAVTPQPRGAE CGACAGCGGCGAGAGAC AGAGCAGCTGGGTCAACAGAGGCG FHMWNYHSHVFSVGDTF TGATGCAGCCCACCCAG TCAACAGAGGCGAGTCCAGCCGGA SLAMHLQYKIHEAPFDL ATGAGCGCCCAGGAAGA AGTCCAGCCGGAAGGCCTACGACC LLEWLYVPIDPTCQPMR TCTGGGCGACGACACCG AGGCCTACGACCACAACAGCCCCT LYSTCLYHPNAPQCLSH GCATCCACGTGATCCCT ACAACAGCCCCTACATCTGGCCCC MNSGCTFTSPHLAQRVA ACCCTGAACGGCGACGA ACATCTGGCCCCGGAACGACTACG STVYQNCEHADNYTAYC CCGGCACAAGATCGTGA GGAACGACTACGACGGCTTCCTGG LGISHMEPSFGLILHDG ACGTGGACCAGCGGCAG ACGGCTTCCTGGAAAATGCCCACG GTTLKFVDTPESLSGLY TACGGCGACGTGTTCAA AAAATGCCCACGAGCACCACGGCG VFVVYFNGHVEAVAYTV GGGCGACCTGAACCCCA AGCACCACGGCGTGTACAACCAGG VSTVDHFVNAIEERGFP AGCCCCAGGGACAGCGG TGTACAACCAGGGCAGAGGCATCG PTAGQPPATTKPKEITP CTGATTGAGGTGTCCGT GCAGAGGCATCGACAGCGGCGAGA VNPGTSPLLRYAAWTGG GGAAGAGAACCACCCCT ACAGCGGCGAGAGACTGATGCAGC LAAVVLLCLVIFLICTA TCACCCTGAGAGCCCCT GACTGATGCAGCCCACCCAGATGA KRMRVKAARVDK ATCCAGCGGATCTACGG CCACCCAGATGA GCGCCCAGGAAGCGTGCGCTATACCGAGA GCGCCCAGGAAG ATCTGGGCGACG CTTGGAGCTTCCTGCCCATCTGGGCGACG ACACCGGCATCC AGCCTGACCTGTACTGG ACACCGGCATCC ACGTGATCCCTACGACGCCGCTCCTGCCA ACGTGATCCCTA CCCTGAACGGCG TCCAGCACATCTGCCTGCCCTGAACGGCG ACGACCGGCACA AAGCACACCACCTGTTT ACGACCGGCACA AGATCGTGAACGCCAGGACGTGGTGGTGG AGATCGTGAACG TGGACCAGCGGC ACGTGGACTGCGCCGAGTGGACCAGCGGC AGTACGGCGACG AACACCAAAGAGGACCA AGTACGGCGACG TGTTCAAGGGCGGCTGGCCGAGATCAGCT TGTTCAAGGGCG ACCTGAACCCCA ACCGGTTCCAGGGCAAGACCTGAACCCCA AGCCCCAGGGAC AAAGAGGCCGACCAGCC AGCCCCAGGGAC AGCGGCTGATTGCTGGATCGTCGTGAACA AGCGGCTGATTG AGGTGTCCGTGG CCAGCACCCTGTTCGACAGGTGTCCGTGG AAGAGAACCACC GAGCTGGAACTGGACCC AAGAGAACCACC CCTTCACCCTGATCCCGAGATCGAACCCG CCTTCACCCTGA GAGCCCCTATCC GGGTGCTGAAGGTGCTGGAGCCCCTATCC AGCGGATCTACG CGGACCGAGAAGCAGTA AGCGGATCTACG GCGTGCGCTATACCTGGGAGTGTACATCT GCGTGCGCTATA CCGAGACTTGGA GGAACATGCGGGGCAGCCCGAGACTTGGA GCTTCCTGCCCA GACGGCACCTCTACCTA GCTTCCTGCCCA GCCTGACCTGTACGCCACCTTCCTCGTGA GCCTGACCTGTA CTGGCGACGCCG CCTGGAAGGGCGACGAGCTGGCGACGCCG CTCCTGCCATCC AAAACCCGGAACCCTAC CTCCTGCCATCC AGCACATCTGCCCCCTGCCGTGACCCCTC AGCACATCTGCC TGAAGCACACCA AGCCTAGAGGCGCCGAGTGAAGCACACCA CCTGTTTCCAGG TTTCACATGTGGAATTA CCTGTTTCCAGG ACGTGGTGGTGGCCACAGCCACGTGTTCA ACGTGGTGGTGG ACGTGGACTGCG GCGTGGGCGACACCTTCACGTGGACTGCG CCGAGAACACCA TCCCTGGCCATGCATCT CCGAGAACACCA AAGAGGACCAGCGCAGTACAAGATCCACG AAGAGGACCAGC TGGCCGAGATCA AGGCCCCTTTCGACCTGTGGCCGAGATCA GCTACCGGTTCC CTGCTGGAATGGCTGTA GCTACCGGTTCC AGGGCAAGAAAGCGTGCCCATCGACCCTA AGGGCAAGAAAG AGGCCGACCAGC CCTGCCAGCCCATGCGGAGGCCGACCAGC CCTGGATCGTCG CTGTACTCCACCTGTCT CCTGGATCGTCG TGAACACCAGCAGTACCACCCCAACGCCC TGAACACCAGCA CCCTGTTCGACG CTCAGTGCCTGAGCCACCCCTGTTCGACG AGCTGGAACTGG ATGAATAGCGGCTGCAC AGCTGGAACTGG ACCCTCCCGAGACTTCACCAGCCCTCACC ACCCTCCCGAGA TCGAACCCGGGG TGGCTCAGAGGGTGGCCTCGAACCCGGGG TGCTGAAGGTGC AGCACCGTGTACCAGAA TGCTGAAGGTGC TGCGGACCGAGATTGCGAGCACGCCGACA TGCGGACCGAGA AGCAGTACCTGG ACTACACCGCCTACTGCAGCAGTACCTGG GAGTGTACATCT CTGGGCATCAGCCACAT GAGTGTACATCT GGAACATGCGGGGGAACCCAGCTTCGGCC GGAACATGCGGG GCAGCGACGGCA TGATCCTGCACGATGGCGCAGCGACGGCA CCTCTACCTACG GGCACCACCCTGAAGTT CCTCTACCTACG CCACCTTCCTCGCGTGGACACCCCTGAGT CCACCTTCCTCG TGACCTGGAAGG CCCTGAGCGGCCTGTACTGACCTGGAAGG GCGACGAGAAAA GTGTTCGTGGTGTACTT GCGACGAGAAAA CCCGGAACCCTACAACGGCCACGTGGAAG CCCGGAACCCTA CCCCTGCCGTGA CCGTGGCCTACACCGTGCCCCTGCCGTGA CCCCTCAGCCTA GTGTCCACCGTGGACCA CCCCTCAGCCTA GAGGCGCCGAGTCTTCGTGAACGCCATCG GAGGCGCCGAGT TTCACATGTGGA AGGAACGGGGCTTCCCTTTCACATGTGGA ATTACCACAGCC CCAACTGCTGGACAGCC ATTACCACAGCC ACGTGTTCAGCGTCCTGCCACCACCAAGC ACGTGTTCAGCG TGGGCGACACCT CCAAAGAAATCACCCCTTGGGCGACACCT TCTCCCTGGCCA GTGAACCCCGGCACCAG TCTCCCTGGCCA TGCATCTGCAGTCCCACTGCTGCGCTATG TGCATCTGCAGT ACAAGATCCACG CTGCTTGGACAGGCGGAACAAGATCCACG AGGCCCCTTTCG CTGGCTGCTGTGGTGCT AGGCCCCTTTCG ACCTGCTGCTGGGCTGTGCCTCGTGATTT ACCTGCTGCTGG AATGGCTGTACG TCCTGATCTGCACCGCCAATGGCTGTACG TGCCCATCGACC AAGCGGATGAGAGTGAA TGCCCATCGACC CTACCTGCCAGCGGCCGCCAGAGTGGACA CTACCTGCCAGC CCATGCGGCTGT AG CCATGCGGCTGT ACTCCACCTGTCACTCCACCTGTC TGTACCACCCCA TGTACCACCCCA ACGCCCCTCAGT ACGCCCCTCAGTGCCTGAGCCACA GCCTGAGCCACA TGAATAGCGGCT TGAATAGCGGCT GCACCTTCACCAGCACCTTCACCA GCCCTCACCTGG GCCCTCACCTGG CTCAGAGGGTGG CTCAGAGGGTGGCCAGCACCGTGT CCAGCACCGTGT ACCAGAATTGCG ACCAGAATTGCG AGCACGCCGACAAGCACGCCGACA ACTACACCGCCT ACTACACCGCCT ACTGCCTGGGCA ACTGCCTGGGCATCAGCCACATGG TCAGCCACATGG AACCCAGCTTCG AACCCAGCTTCG GCCTGATCCTGCGCCTGATCCTGC ACGATGGCGGCA ACGATGGCGGCA CCACCCTGAAGT CCACCCTGAAGTTCGTGGACACCC TCGTGGACACCC CTGAGTCCCTGA CTGAGTCCCTGA GCGGCCTGTACGGCGGCCTGTACG TGTTCGTGGTGT TGTTCGTGGTGT ACTTCAACGGCC ACTTCAACGGCCACGTGGAAGCCG ACGTGGAAGCCG TGGCCTACACCG TGGCCTACACCG TGGTGTCCACCGTGGTGTCCACCG TGGACCACTTCG TGGACCACTTCG TGAACGCCATCG TGAACGCCATCGAGGAACGGGGCT AGGAACGGGGCT TCCCTCCAACTG TCCCTCCAACTG CTGGACAGCCTCCTGGACAGCCTC CTGCCACCACCA CTGCCACCACCA AGCCCAAAGAAA AGCCCAAAGAAATCACCCCTGTGA TCACCCCTGTGA ACCCCGGCACCA ACCCCGGCACCA GCCCACTGCTGCGCCCACTGCTGC GCTATGCTGCTT GCTATGCTGCTT GGACAGGCGGAC GGACAGGCGGACTGGCTGCTGTGG TGGCTGCTGTGG TGCTGCTGTGCC TGCTGCTGTGCC TCGTGATTTTCCTCGTGATTTTCC TGATCTGCACCG TGATCTGCACCG CCAAGCGGATGA CCAAGCGGATGAGAGTGAAGGCCG GAGTGAAGGCCG CCAGAGTGGACA CCAGAGTGGACA AGTGATAATAGGAGTGATAATAGG CTGGAGCCTCGG CTGGAGCCTCGG TGGCCATGCTTC TGGCCATGCTTCTTGCCCCTTGGG TTGCCCCTTGGG CCTCCCCCCAGC CCTCCCCCCAGC CCCTCCTCCCCTCCCTCCTCCCCT TCCTGCACCCGT TCCTGCACCCGT ACCCCCGTGGTC ACCCCCGTGGTCTTTGAATAAAGT TTTGAATAAAGT CTGAGTGGGCGG CTGAGTGGGCGG C CAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAA AAAAATCTAG SEQ ID NO: 64 SEQ ID NO: 65SEQ ID NO: 66 SEQ ID NO: 67 VZV-GE- GGGAAATAAGAG MGTVNKPVVGVLMGFGIATGGGGACAGTTAATAA GGGAAATAAGAG truncated- AGAAAAGAAGAG ITGTLRITNPVRASVLRACCTGTGGTGGGGGTAT AGAAAAGAAGAG delete_ TAAGAAGAAATA YDDFHIDEDKLDTNSVYTGATGGGGTTCGGAATT TAAGAAGAAATA from_574_-_ TAAGAGCCACCAEPYYHSDHAESSWVNRG ATCACGGGAACGTTGCG TAAGAGCCACCA Y569A TGGGGACAGTTAESSRKAYDHNSPYIWPR TATAACGAATCCGGTCA TGGGGACAGTTA Variant ATAAACCTGTGGNDYDGFLENAHEHHGVY GAGCATCCGTCTTGCGA ATAAACCTGTGG 2 TGGGGGTATTGANQGRGIDSGERLMQPTQ TACGATGATTTTCACAT TGGGGGTATTGA TGGGGTTCGGAAMSAQEDLGDDTGIHVIP CGATGAAGACAAACTGG TGGGGTTCGGAA TTATCACGGGAATLNGDDRHKIVNVDQRQ ATACAAACTCCGTATAT TTATCACGGGAA CGTTGCGTATAAYGDVFKGDLNPKPQGQR GAGCCTTACTACCATTC CGTTGCGTATAA CGAATCCGGTCALIEVSVEENHPFTLRAP AGATCATGCGGAGTCTT CGAATCCGGTCA GAGCATCCGTCTIQRIYGVRYTETWSFLP CATGGGTAAATCGGGGA GAGCATCCGTCT TGCGATACGATGSLTCTGDAAPAIQHICL GAGTCTTCGCGAAAAGC TGCGATACGATG ATTTTCACATCGKHTTCFQDVVVDVDCAE GTACGATCATAACTCAC ATTTTCACATCG ATGAAGACAAACNTKEDQLAEISYRFQGK CTTATATATGGCCACGT ATGAAGACAAAC TGGATACAAACTKEADQPWIVVNTSTLFD AATGATTATGATGGATT TGGATACAAACT CCGTATATGAGCELELDPPEIEPGVLKVL TTTAGAGAACGCACACG CCGTATATGAGC CTTACTACCATTRTEKQYLGVYIWNMRGS AACACCATGGGGTGTAT CTTACTACCATT CAGATCATGCGGDGTSTYATFLVTWKGDE AATCAGGGCCGTGGTAT CAGATCATGCGG AGTCTTCATGGGKTRNPTPAVTPQPRGAE CGATAGCGGGGAACGGT AGTCTTCATGGG TAAATCGGGGAGFHMWNYHSHVFSVGDTF TAATGCAACCCACACAA TAAATCGGGGAG AGTCTTCGCGAASLAMHLQYKIHEAPFDL ATGTCTGCACAGGAGGA AGTCTTCGCGAA AAGCGTACGATCLLEWLYVPIDPTCQPMR TCTTGGGGACGATACGG AAGCGTACGATC ATAACTCACCTTLYSTCLYHPNAPQCLSH GCATCCACGTTATCCCT ATAACTCACCTT ATATATGGCCACMNSGCTFTSPHLAQRVA ACGTTAAACGGCGATGA ATATATGGCCAC GTAATGATTATGSTVYQNCEHADNYTAYC CAGACATAAAATTGTAA GTAATGATTATG ATGGATTTTTAGLGISHMEPSFGLILHDG ATGTGGACCAACGTCAA ATGGATTTTTAG AGAACGCACACGGTTLKFVDTPESLSGLY TACGGTGACGTGTTTAA AGAACGCACACG AACACCATGGGGVFVVYFNGHVEAVAYTV AGGAGATCTTAATCCAA AACACCATGGGG TGTATAATCAGGVSTVDHFVNAIEERGFP AACCCCAAGGCCAAAGA TGTATAATCAGG GCCGTGGTATCGPTAGQPPATTKPKEITP CTCATTGAGGTGTCAGT GCCGTGGTATCG ATAGCGGGGAACVNPGTSPLLRYAAWTGG GGAAGAAAATCACCCGT ATAGCGGGGAAC GGTTAATGCAACLAAVVLLCLVIFLICTA TTACTTTACGCGCACCG GGTTAATGCAAC CCACACAAATGTKRMRVKAARVDK ATTCAGCGGATTTATGG CCACACAAATGT CTGCACAGGAGGAGTCCGGTACACCGAGA CTGCACAGGAGG ATCTTGGGGACG CTTGGAGCTTTTTGCCGATCTTGGGGACG ATACGGGCATCC TCATTAACCTGTACGGG ATACGGGCATCC ACGTTATCCCTAAGACGCAGCGCCCGCCA ACGTTATCCCTA CGTTAAACGGCG TCCAGCATATATGTTTACGTTAAACGGCG ATGACAGACATA AAACATACAACATGCTT ATGACAGACATA AAATTGTAAATGTCAAGACGTGGTGGTGG AAATTGTAAATG TGGACCAACGTC ATGTGGATTGCGCGGAATGGACCAACGTC AATACGGTGACG AATACTAAAGAGGATCA AATACGGTGACG TGTTTAAAGGAGGTTGGCCGAAATCAGTT TGTTTAAAGGAG ATCTTAATCCAA ACCGTTTTCAAGGTAAGATCTTAATCCAA AACCCCAAGGCC AAGGAAGCGGACCAACC AACCCCAAGGCC AAAGACTCATTGGTGGATTGTTGTAAACA AAAGACTCATTG AGGTGTCAGTGG CGAGCACACTGTTTGATAGGTGTCAGTGG AAGAAAATCACC GAACTCGAATTAGACCC AAGAAAATCACC CGTTTACTTTACCCCCGAGATTGAACCGG CGTTTACTTTAC GCGCACCGATTC GTGTCTTGAAAGTACTTGCGCACCGATTC AGCGGATTTATG CGGACAGAGAAACAATA AGCGGATTTATG GAGTCCGGTACACTTGGGTGTGTACATTT GAGTCCGGTACA CCGAGACTTGGA GGAACATGCGCGGCTCCCCGAGACTTGGA GCTTTTTGCCGT GATGGTACGTCTACCTA GCTTTTTGCCGT CATTAACCTGTACGCCACGTTTTTGGTCA CATTAACCTGTA CGGGAGACGCAG CCTGGAAAGGGGATGAGCGGGAGACGCAG CGCCCGCCATCC AAGACAAGAAACCCTAC CGCCCGCCATCC AGCATATATGTTGCCCGCAGTAACTCCTC AGCATATATGTT TAAAACATACAA AACCAAGAGGGGCTGAGTAAAACATACAA CATGCTTTCAAG TTTCATATGTGGAATTA CATGCTTTCAAG ACGTGGTGGTGGCCACTCGCATGTATTTT ACGTGGTGGTGG ATGTGGATTGCG CAGTTGGTGATACGTTTATGTGGATTGCG CGGAAAATACTA AGCTTGGCAATGCATCT CGGAAAATACTA AAGAGGATCAGTTCAGTATAAGATACATG AAGAGGATCAGT TGGCCGAAATCA AAGCGCCATTTGATTTGTGGCCGAAATCA GTTACCGTTTTC CTGTTAGAGTGGTTGTA GTTACCGTTTTC AAGGTAAGAAGGTGTCCCCATCGATCCTA AAGGTAAGAAGG AAGCGGACCAAC CATGTCAACCAATGCGGAAGCGGACCAAC CGTGGATTGTTG TTATATTCTACGTGTTT CGTGGATTGTTG TAAACACGAGCAGTATCATCCCAACGCAC TAAACACGAGCA CACTGTTTGATG CCCAATGCCTCTCTCATCACTGTTTGATG AACTCGAATTAG ATGAATTCCGGTTGTAC AACTCGAATTAG ACCCCCCCGAGAATTTACCTCGCCACATT ACCCCCCCGAGA TTGAACCGGGTG TAGCCCAGCGTGTTGCATTGAACCGGGTG TCTTGAAAGTAC AGCACAGTGTATCAAAA TCTTGAAAGTAC TTCGGACAGAGATTGTGAACATGCAGATA TTCGGACAGAGA AACAATACTTGG ACTACACCGCATATTGTAACAATACTTGG GTGTGTACATTT CTGGGAATATCTCATAT GTGTGTACATTT GGAACATGCGCGGGAGCCTAGCTTTGGTC GGAACATGCGCG GCTCCGATGGTA TAATCTTACACGACGGGGCTCCGATGGTA CGTCTACCTACG GGCACCACGTTAAAGTT CGTCTACCTACG CCACGTTTTTGGTGTAGATACACCCGAGA CCACGTTTTTGG TCACCTGGAAAG GTTTGTCGGGATTATACTCACCTGGAAAG GGGATGAGAAGA GTTTTTGTGGTGTATTT GGGATGAGAAGA CAAGAAACCCTATAACGGGCATGTTGAAG CAAGAAACCCTA CGCCCGCAGTAA CCGTAGCATACACTGTTCGCCCGCAGTAA CTCCTCAACCAA GTATCCACAGTAGATCA CTCCTCAACCAA GAGGGGCTGAGTTTTTGTAAACGCAATTG GAGGGGCTGAGT TTCATATGTGGA AAGAGCGTGGATTTCCGTTCATATGTGGA ATTACCACTCGC CCAACGGCCGGTCAGCC ATTACCACTCGC ATGTATTTTCAGACCGGCGACTACTAAAC ATGTATTTTCAG TTGGTGATACGT CCAAGGAAATTACCCCCTTGGTGATACGT TTAGCTTGGCAA GTAAACCCCGGAACGTC TTAGCTTGGCAA TGCATCTTCAGTACCACTTCTACGATATG TGCATCTTCAGT ATAAGATACATG CCGCATGGACCGGAGGGATAAGATACATG AAGCGCCATTTG CTTGCAGCAGTAGTACT AAGCGCCATTTG ATTTGCTGTTAGTTTATGTCTCGTAATAT ATTTGCTGTTAG AGTGGTTGTATG TTTTAATCTGTACGGCTAGTGGTTGTATG TCCCCATCGATC AAACGAATGAGGGTTAA TCCCCATCGATC CTACATGTCAACAGCCGCCAGGGTAGACA CTACATGTCAAC CAATGCGGTTAT AG CAATGCGGTTAT ATTCTACGTGTTATTCTACGTGTT TGTATCATCCCA TGTATCATCCCA ACGCACCCCAAT ACGCACCCCAATGCCTCTCTCATA GCCTCTCTCATA TGAATTCCGGTT TGAATTCCGGTT GTACATTTACCTGTACATTTACCT CGCCACATTTAG CGCCACATTTAG CCCAGCGTGTTG CCCAGCGTGTTGCAAGCACAGTGT CAAGCACAGTGT ATCAAAATTGTG ATCAAAATTGTG AACATGCAGATAAACATGCAGATA ACTACACCGCAT ACTACACCGCAT ATTGTCTGGGAA ATTGTCTGGGAATATCTCATATGG TATCTCATATGG AGCCTAGCTTTG AGCCTAGCTTTG GTCTAATCTTACGTCTAATCTTAC ACGACGGGGGCA ACGACGGGGGCA CCACGTTAAAGT CCACGTTAAAGTTTGTAGATACAC TTGTAGATACAC CCGAGAGTTTGT CCGAGAGTTTGT CGGGATTATACGCGGGATTATACG TTTTTGTGGTGT TTTTTGTGGTGT ATTTTAACGGGC ATTTTAACGGGCATGTTGAAGCCG ATGTTGAAGCCG TAGCATACACTG TAGCATACACTG TTGTATCCACAGTTGTATCCACAG TAGATCATTTTG TAGATCATTTTG TAAACGCAATTG TAAACGCAATTGAAGAGCGTGGAT AAGAGCGTGGAT TTCCGCCAACGG TTCCGCCAACGG CCGGTCAGCCACCCGGTCAGCCAC CGGCGACTACTA CGGCGACTACTA AACCCAAGGAAA AACCCAAGGAAATTACCCCCGTAA TTACCCCCGTAA ACCCCGGAACGT ACCCCGGAACGT CACCACTTCTACCACCACTTCTAC GATATGCCGCAT GATATGCCGCAT GGACCGGAGGGC GGACCGGAGGGCTTGCAGCAGTAG TTGCAGCAGTAG TACTTTTATGTC TACTTTTATGTC TCGTAATATTTTTCGTAATATTTT TAATCTGTACGG TAATCTGTACGG CTAAACGAATGA CTAAACGAATGAGGGTTAAAGCCG GGGTTAAAGCCG CCAGGGTAGACA CCAGGGTAGACA AGTGATAATAGGAGTGATAATAGG CTGGAGCCTCGG CTGGAGCCTCGG TGGCCATGCTTC TGGCCATGCTTCTTGCCCCTTGGG TTGCCCCTTGGG CCTCCCCCCAGC CCTCCCCCCAGC CCCTCCTCCCCTCCCTCCTCCCCT TCCTGCACCCGT TCCTGCACCCGT ACCCCCGTGGTC ACCCCCGTGGTCTTTGAATAAAGT TTTGAATAAAGT CTGAGTGGGCGG CTGAGTGGGCGG C CAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAA AAAAATCTAG SEQ ID NO: 68 SEQ ID NO: 69SEQ ID NO: 70 SEQ ID NO: 71 VZV-GE- GGGAAATAAGAG MGTVNKPVVGVLMGFGIATGGGGACAGTTAATAA GGGAAATAAGAG truncated- AGAAAAGAAGAG ITGTLRITNPVRASVLRACCTGTGGTGGGGGTAT AGAAAAGAAGAG delete_ TAAGAAGAAATA YDDFHIDEDKLDTNSVYTGATGGGGTTCGGAATT TAAGAAGAAATA from_574_-_ TAAGAGCCACCAEPYYHSDHAESSWVNRG ATCACGGGAACGTTGCG TAAGAGCCACCA Y569A TGGGGACAGTTAESSRKAYDHNSPYIWPR TATAACGAATCCGGTCA TGGGGACAGTTA Variant ATAAACCTGTGGNDYDGFLENAHEHHGVY GAGCATCCGTCTTGCGA ATAAACCTGTGG 3 TGGGGGTATTGANQGRGIDSGERLMQPTQ TACGATGATTTTCACAT TGGGGGTATTGA TGGGGTTCGGAAMSAQEDLGDDTGIHVIP CGATGAAGACAAACTGG TGGGGTTCGGAA TTATCACGGGAATLNGDDRHKIVNVDQRQ ATACAAACTCCGTATAT TTATCACGGGAA CGTTGCGTATAAYGDVFKGDLNPKPQGQR GAGCCTTACTACCATTC CGTTGCGTATAA CGAATCCGGTCALIEVSVEENHPFTLRAP AGATCATGCGGAGTCTT CGAATCCGGTCA GAGCATCCGTCTIQRIYGVRYTETWSFLP CATGGGTAAATCGGGGA GAGCATCCGTCT TGCGATACGATGSLTCTGDAAPAIQHICL GAGTCTTCGCGAAAAGC TGCGATACGATG ATTTTCACATCGKHTTCFQDVVVDVDCAE GTACGATCATAACTCAC ATTTTCACATCG ATGAAGACAAACNTKEDQLAEISYRFQGK CTTATATATGGCCACGT ATGAAGACAAAC TGGATACAAACTKEADQPWIVVNTSTLFD AATGATTATGATGGATT TGGATACAAACT CCGTATATGAGCELELDPPEIEPGVLKVL TTTAGAGAACGCACACG CCGTATATGAGC CTTACTACCATTRTEKQYLGVYIWNMRGS AACACCATGGGGTGTAT CTTACTACCATT CAGATCATGCGGDGTSTYATFLVTWKGDE AATCAGGGCCGTGGTAT CAGATCATGCGG AGTCTTCATGGGKTRNPTPAVTPQPRGAE CGATAGCGGGGAACGGT AGTCTTCATGGG TAAATCGGGGAGFHMWNYHSHVFSVGDTF TAATGCAACCCACACAA TAAATCGGGGAG AGTCTTCGCGAASLAMHLQYKIHEAPFDL ATGTCTGCACAGGAGGA AGTCTTCGCGAA AAGCGTACGATCLLEWLYVPIDPTCQPMR TCTTGGGGACGATACGG AAGCGTACGATC ATAACTCACCTTLYSTCLYHPNAPQCLSH GCATCCACGTTATCCCT ATAACTCACCTT ATATATGGCCACMNSGCTFTSPHLAQRVA ACGTTAAACGGCGATGA ATATATGGCCAC GTAATGATTATGSTVYQNCEHADNYTAYC CAGACATAAAATTGTAA GTAATGATTATG ATGGATTTTTAGLGISHMEPSFGLILHDG ATGTGGACCAACGTCAA ATGGATTTTTAG AGAACGCACACGGTTLKFVDTPESLSGLY TACGGTGACGTGTTTAA AGAACGCACACG AACACCATGGGGVFVVYFNGHVEAVAYTV AGGAGATCTTAATCCAA AACACCATGGGG TGTATAATCAGGVSTVDHFVNAIEERGFP AACCCCAAGGCCAAAGA TGTATAATCAGG GCCGTGGTATCGPTAGQPPATTKPKEITP CTCATTGAGGTGTCAGT GCCGTGGTATCG ATAGCGGGGAACVNPGTSPLLRYAAWTGG GGAAGAAAATCACCCGT ATAGCGGGGAAC GGTTAATGCAACLAAVVLLCLVIFLICTA TTACTTTACGCGCACCG GGTTAATGCAAC CCACACAAATGTKRMRVKAARVDK ATTCAGCGGATTTATGG CCACACAAATGT CTGCACAGGAGGAGTCCGGTACACCGAGA CTGCACAGGAGG ATCTTGGGGACG CTTGGAGCTTTTTGCCGATCTTGGGGACG ATACGGGCATCC TCATTAACCTGTACGGG ATACGGGCATCC ACGTTATCCCTAAGACGCAGCGCCCGCCA ACGTTATCCCTA CGTTAAACGGCG TCCAGCATATATGTTTACGTTAAACGGCG ATGACAGACATA AAACATACAACATGCTT ATGACAGACATA AAATTGTAAATGTCAAGACGTGGTGGTGG AAATTGHAAATG TGGACCAACGTC ATGTGGATTGCGCGGAATGGACCAACGTC AATACGGTGACG AATACTAAAGAGGATCA AATACGGTGACG TGTTTAAAGGAGGTTGGCCGAAATCAGTT TGTTTAAAGGAG ATCTTAATCCAA ACCGTTTTCAAGGTAAGATCTTAATCCAA AACCCCAAGGCC AAGGAAGCGGACCAACC AACCCCAAGGCC AAAGACTCATTGGTGGATTGTTGTAAACA AAAGACTCATTG AGGTGTCAGTGG CGAGCACACTGTTTGATAGGTGTCAGTGG AAGAAAATCACC GAACTCGAATTAGACCC AAGAAAATCACC CGTTTACTTTACACCCGAGATTGAACCGG CGTTTACTTTAC GCGCACCGATTC GTGTCTTGAAAGTACTTGCGCACCGATTC AGCGGATTTATG CGGACAGAGAAACAATA AGCGGATTTATG GAGTCCGGTACACTTGGGTGTGTACATTT GAGTCCGGTACA CCGAGACTTGGA GGAACATGCGCGGCTCCCCGAGACTTGGA GCTTTTTGCCGT GATGGTACGTCTACCTA GCTTTTTGCCGT CATTAACCTGTACGCCACGTTTTTGGTCA CATTAACCTGTA CGGGAGACGCAG CCTGGAAAGGGGATGAGCGGGAGACGCAG CGCCCGCCATCC AAGACAAGAAACCCTAC CGCCCGCCATCC AGCATATATGTTGCCCGCAGTAACTCCTC AGCATATATGTT TAAAACATACAA AACCAAGAGGGGCTGAGTAAAACATACAA CATGCTTTCAAG TTTCATATGTGGAATTA CATGCTTTCAAG ACGTGGTGGTGGCCACTCGCATGTATTTT ACGTGGTGGTGG ATGTGGATTGCG CAGTTGGTGATACGTTTATGTGGATTGCG CGGAAAATACTA AGCTTGGCAATGCATCT CGGAAAATACTA AAGAGGATCAGTTCAGTATAAGATACATG AAGAGGATCAGT TGGCCGAAATCA AAGCGCCATTTGATTTGTGGCCGAAATCA GTTACCGTTTTC CTGTTAGAGTGGTTGTA GTTACCGTTTTC AAGGTAAGAAGGTGTCCCCATCGATCCTA AAGGTAAGAAGG AAGCGGACCAAC CATGTCAACCAATGCGGAAGCGGACCAAC CGTGGATTGTTG TTATATTCTACGTGTTT CGTGGATTGTTG TAAACACGAGCAGTATCATCCCAACGCAC TAAACACGAGCA CACTGTTTGATG CCCAATGCCTCTCTCATCACTGTTTGATG AACTCGAATTAG ATGAATTCCGGTTGTAC AACTCGAATTAG ACCCACCCGAGAATTTACCTCGCCACATT ACCCACCCGAGA TTGAACCGGGTG TAGCCCAGCGTGTTGCATTGAACCGGGTG TCTTGAAAGTAC AGCACAGTGTATCAAAA TCTTGAAAGTAC TTCGGACAGAGATTGTGAACATGCAGATA TTCGGACAGAGA AACAATACTTGG ACTACACCGCATATTGTAACAATACTTGG GTGTGTACATTT CTGGGAATATCTCATAT GTGTGTACATTT GGAACATGCGCGGGAGCCTAGCTTTGGTC GGAACATGCGCG GCTCCGATGGTA TAATCTTACACGACGGGGCTCCGATGGTA CGTCTACCTACG GGCACCACGTTAAAGTT CGTCTACCTACG CCACGTTTTTGGTGTAGATACACCCGAGA CCACGTTTTTGG TCACCTGGAAAG GTTTGTCGGGATTATACTCACCTGGAAAG GGGATGAGAAGA GTTTTTGTGGTGTATTT GGGATGAGAAGA CAAGAAACCCTATAACGGGCATGTTGAAG CAAGAAACCCTA CGCCCGCAGTAA CCGTAGCATACACTGTTCGCCCGCAGTAA CTCCTCAACCAA GTATCCACAGTAGATCA CTCCTCAACCAA GAGGGGCTGAGTTTTTGTAAACGCAATTG GAGGGGCTGAGT TTCATATGTGGA AAGAGCGTGGATTTCCGTTCATATGTGGA ATTACCACTCGC CCAACGGCCGGTCAGCC ATTACCACTCGC ATGTATTTTCAGACCGGCGACTACTAAAC ATGTATTTTCAG TTGGTGATACGT CCAAGGAAATTACCCCCTTGGTGATACGT TTAGCTTGGCAA GTAAACCCCGGAACGTC TTAGCTTGGCAA TGCATCTTCAGTACCACTTCTACGATATG TGCATCTTCAGT ATAAGATACATG CCGCATGGACCGGAGGGATAAGATACATG AAGCGCCATTTG CTTGCAGCAGTAGTACT AAGCGCCATTTG ATTTGCTGTTAGTTTATGTCTCGTAATAT ATTTGCTGTTAG AGTGGTTGTATG TTTTAATCTGTACGGCTAGTGGTTGTATG TCCCCATCGATC AAACGAATGAGGGTTAA TCCCCATCGATC CTACATGTCAACAGCCGCCAGGGTAGACA CTACATGTCAAC CAATGCGGTTAT AG CAATGCGGTTAT ATTCTACGTGTTATTCTACGTGTT TGTATCATCCCA TGTATCATCCCA ACGCACCCCAAT ACGCACCCCAATGCCTCTCTCATA GCCTCTCTCATA TGAATTCCGGTT TGAATTCCGGTT GTACATTTACCTGTACATTTACCT CGCCACATTTAG CGCCACATTTAG CCCAGCGTGTTG CCCAGCGTGTTGCAAGCACAGTGT CAAGCACAGTGT ATCAAAATTGTG ATCAAAATTGTG AACATGCAGATAAACATGCAGATA ACTACACCGCAT ACTACACCGCAT ATTGTCTGGGAA ATTGTCTGGGAATATCTCATATGG TATCTCATATGG AGCCTAGCTTTG AGCCTAGCTTTG GTCTAATCTTACGTCTAATCTTAC ACGACGGGGGCA ACGACGGGGGCA CCACGTTAAAGT CCACGTTAAAGTTTGTAGATACAC TTGTAGATACAC CCGAGAGTTTGT CCGAGAGTTTGT CGGGATTATACGCGGGATTATACG TTTTTGTGGTGT TTTTTGTGGTGT ATTTTAACGGGC ATTTTAACGGGCATGTTGAAGCCG ATGTTGAAGCCG TAGCATACACTG TAGCATACACTG TTGTATCCACAGTTGTATCCACAG TAGATCATTTTG TAGATCATTTTG TAAACGCAATTG TAAACGCAATTGAAGAGCGTGGAT AAGAGCGTGGAT TTCCGCCAACGG TTCCGCCAACGG CCGGTCAGCCACCCGGTCAGCCAC CGGCGACTACTA CGGCGACTACTA AACCCAAGGAAA AACCCAAGGAAATTACCCCCGTAA TTACCCCCGTAA ACCCCGGAACGT ACCCCGGAACGT CACCACTTCTACCACCACTTCTAC GATATGCCGCAT GATATGCCGCAT GGACCGGAGGGC GGACCGGAGGGCTTGCAGCAGTAG TTGCAGCAGTAG TACTTTTATGTC TACTTTTATGTC TCGTAATATTTTTCGTAATATTTT TAATCTGTACGG TAATCTGTACGG CTAAACGAATGA CTAAACGAATGAGGGTTAAAGCCG GGGTTAAAGCCG CCAGGGTAGACA CCAGGGTAGACA AGTGATAATAGGAGTGATAATAGG CTGGAGCCTCGG CTGGAGCCTCGG TGGCCATGCTTC TGGCCATGCTTCTTGCCCCTTGGG TTGCCCCTTGGG CCTCCCCCCAGC CCTCCCCCCAGC CCCTCCTCCCCTCCCTCCTCCCCT TCCTGCACCCGT TCCTGCACCCGT ACCCCCGTGGTC ACCCCCGTGGTCTTTGAATAAAGT TTTGAATAAAGT CTGAGTGGGCGG CTGAGTGGGCGG C CAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAA AAAAATCTAG SEQ ID NO: 72 SEQ ID NO: 73SEQ ID NO: 74 SEQ ID NO: 75 VZV-GE- GGGAAATAAGAG MGTVNKPVVGVLMGFGIATGGGGACAGTTAATAA GGGAAATAAGAG truncated- AGAAAAGAAGAG ITGTLRITNPVRASVLRACCTGTGGTGGGGGTAT AGAAAAGAAGAG delete_ TAAGAAGAAATA YDDFHIDEDKLDTNSVYTGATGGGGTTCGGAATT TAAGAAGAAATA from_574_-_ TAAGAGCCACCAEPYYHSDHAESSWVNRG ATCACGGGAACGTTGCG TAAGAGCCACCA Y569A TGGGGACAGTTAESSRKAYDHNSPYIWPR TATAACGAATCCGGTCA TGGGGACAGTTA Variant ATAAACCTGTGGNDYDGFLENAHEHHGVY GAGCATCCGTCTTGCGA ATAAACCTGTGG 4 TGGGGGTATTGANQGRGIDSGERLMQPTQ TACGATGATTTTCACAT TGGGGGTATTGA TGGGGTTCGGAAMSAQEDLGDDTGIHVIP CGATGAAGACAAACTGG TGGGGTTCGGAA TTATCACGGGAATLNGDDRHKIVNVDQRQ ATACAAACTCCGTATAT TTATCACGGGAA CGTTGCGTATAAYGDVFKGDLNPKPQGQR GAGCCTTACTACCATTC CGTTGCGTATAA CGAATCCGGTCALIEVSVEENHPFTLRAP AGATCATGCGGAGTCTT CGAATCCGGTCA GAGCATCCGTCTIQRIYGVRYTETWSFLP CATGGGTAAATCGGGGA GAGCATCCGTCT TGCGATACGATGSLTCTGDAAPAIQHICL GAGTCTTCGCGAAAGGC TGCGATACGATG ATTTTCACATCGKHTTCFQDVVVDVDCAE GTACGATCATAACTCAC ATTTTCACATCG ATGAAGACAAACNTKEDQLAEISYRFQGK CTTATATATGGCCACGT ATGAAGACAAAC TGGATACAAACTKEADQPWIVVNTSTLFD AATGATTATGATGGATT TGGATACAAACT CCGTATATGAGCELELDPPEIEPGVLKVL TTTAGAGAACGCACACG CCGTATATGAGC CTTACTACCATTRTEKQYLGVYIWNMRGS AACACCATGGGGTGTAT CTTACTACCATT CAGATCATGCGGDGTSTYATFLVTWKGDE AATCAGGGCCGTGGTAT CAGATCATGCGG AGTCTTCATGGGKTRNPTPAVTPQPRGAE CGATAGCGGGGAACGGT AGTCTTCATGGG TAAATCGGGGAGFHMWNYHSHVFSVGDTF TAATGCAACCCACACAA TAAATCGGGGAG AGTCTTCGCGAASLAMHLQYKIHEAPFDL ATGTCTGCACAGGAGGA AGTCTTCGCGAA AGGCGTACGATCLLEWLYVPIDPTCQPMR TCTTGGGGACGATACGG AGGCGTACGATC ATAACTCACCTTLYSTCLYHPNAPQCLSH GCATCCACGTTATCCCT ATAACTCACCTT ATATATGGCCACMNSGCTFTSPHLAQRVA ACGTTAAACGGCGATGA ATATATGGCCAC GTAATGATTATGSTVYQNCEHADNYTAYC CAGACATAAGATTGTAA GTAATGATTATG ATGGATTTTTAGLGISHMEPSFGLILHDG ATGTGGACCAACGTCAA ATGGATTTTTAG AGAACGCACACGGTTLKFVDTPESLSGLY TACGGTGACGTGTTTAA AGAACGCACACG AACACCATGGGGVFVVYFNGHVEAVAYTV AGGAGATCTTAATCCAA AACACCATGGGG TGTATAATCAGGVSTVDHFVNAIEERGFP AGCCCCAAGGCCAAAGA TGTATAATCAGG GCCGTGGTATCGPTAGQPPATTKPKEITP CTCATTGAGGTGTCAGT GCCGTGGTATCG ATAGCGGGGAACVNPGTSPLLRYAAWTGG GGAAGAGAATCACCCGT ATAGCGGGGAAC GGTTAATGCAACLAAVVLLCLVIFLICTA TTACTTTACGCGCACCG GGTTAATGCAAC CCACACAAATGTKRMRVKAARVDK ATTCAGCGGATTTATGG CCACACAAATGT CTGCACAGGAGGAGTCCGGTACACCGAGA CTGCACAGGAGG ATCTTGGGGACG CTTGGAGCTTTTTGCCGATCTTGGGGACG ATACGGGCATCC TCATTAACCTGTACGGG ATACGGGCATCC ACGTTATCCCTAAGACGCAGCGCCCGCCA ACGTTATCCCTA CGTTAAACGGCG TCCAGCATATATGTTTACGTTAAACGGCG ATGACAGACATA AAGCATACAACATGCTT ATGACAGACATA AGATTGTAAATGTCAAGACGTGGTGGTGG AGATTGTAAATG TGGACCAACGTC ATGTGGATTGCGCGGAGTGGACCAACGTC AATACGGTGACG AATACTAAAGAGGATCA AATACGGTGACG TGTTTAAAGGAGGTTGGCCGAAATCAGTT TGTTTAAAGGAG ATCTTAATCCAA ACCGTTTTCAAGGTAAGATCTTAATCCAA AGCCCCAAGGCC AAGGAAGCGGACCAACC AGCCCCAAGGCC AAAGACTCATTGGTGGATTGTTGTAAACA AAAGACTCATTG AGGTGTCAGTGG CGAGCACACTGTTTGATAGGTGTCAGTGG AAGAGAATCACC GAACTCGAATTAGACCC AAGAGAATCACC CGTTTACTTTACCCCCGAGATTGAACCGG CGTTTACTTTAC GCGCACCGATTC GTGTCTTGAAAGTACTTGCGCACCGATTC AGCGGATTTATG CGGACAGAGAAACAATA AGCGGATTTATG GAGTCCGGTACACTTGGGTGTGTACATTT GAGTCCGGTACA CCGAGACTTGGA GGAACATGCGCGGCTCCCCGAGACTTGGA GCTTTTTGCCGT GATGGTACGTCTACCTA GCTTTTTGCCGT CATTAACCTGTACGCCACGTTTTTGGTCA CATTAACCTGTA CGGGAGACGCAG CCTGGAAAGGGGATGAGCGGGAGACGCAG CGCCCGCCATCC AAGACAAGAAACCCTAC CGCCCGCCATCC AGCATATATGTTGCCCGCAGTAACTCCTC AGCATATATGTT TAAAGCATACAA AACCAAGAGGGGCTGAGTAAAGCATACAA CATGCTTTCAAG TTTCATATGTGGAATTA CATGCTTTCAAG ACGTGGTGGTGGCCACTCGCATGTATTTT ACGTGGTGGTGG ATGTGGATTGCG CAGTTGGTGATACGTTTATGTGGATTGCG CGGAGAATACTA AGCTTGGCAATGCATCT CGGAGAATACTA AAGAGGATCAGTTCAGTATAAGATACATG AAGAGGATCAGT TGGCCGAAATCA AAGCGCCATTTGATTTGTGGCCGAAATCA GTTACCGTTTTC CTGTTAGAGTGGTTGTA GTTACCGTTTTC AAGGTAAGAAGGTGTCCCCATCGATCCTA AAGGTAAGAAGG AAGCGGACCAAC CATGTCAACCAATGCGGAAGCGGACCAAC CGTGGATTGTTG TTATATTCTACGTGTTT CGTGGATTGTTG TAAACACGAGCAGTATCATCCCAACGCAC TAAACACGAGCA CACTGTTTGATG CCCAATGCCTCTCTCATCACTGTTTGATG AACTCGAATTAG ATGAATTCCGGTTGTAC AACTCGAATTAG ACCCCCCCGAGAATTTACCTCGCCACATT ACCCCCCCGAGA TTGAACCGGGTG TAGCCCAGCGTGTTGCATTGAACCGGGTG TCTTGAAAGTAC AGCACAGTGTATCAGAA TCTTGAAAGTAC TTCGGACAGAGATTGTGAACATGCAGATA TTCGGACAGAGA AACAATACTTGG ACTACACCGCATATTGTAACAATACTTGG GTGTGTACATTT CTGGGAATATCTCATAT GTGTGTACATTT GGAACATGCGCGGGAGCCTAGCTTTGGTC GGAACATGCGCG GCTCCGATGGTA TAATCTTACACGACGGGGCTCCGATGGTA CGTCTACCTACG GGCACCACGTTAAAGTT CGTCTACCTACG CCACGTTTTTGGTGTAGATACACCCGAGA CCACGTTTTTGG TCACCTGGAAAG GTTTGTCGGGATTATACTCACCTGGAAAG GGGATGAGAAGA GTTTTTGTGGTGTATTT GGGATGAGAAGA CAAGAAACCCTATAACGGGCATGTTGAAG CAAGAAACCCTA CGCCCGCAGTAA CCGTAGCATACACTGTTCGCCCGCAGTAA CTCCTCAACCAA GTATCCACAGTAGATCA CTCCTCAACCAA GAGGGGCTGAGTTTTTGTAAACGCAATTG GAGGGGCTGAGT TTCATATGTGGA AAGAGCGTGGATTTCCGTTCATATGTGGA ATTACCACTCGC CCAACGGCCGGTCAGCC ATTACCACTCGC ATGTATTTTCAGACCGGCGACTACTAAAC ATGTATTTTCAG TTGGTGATACGT CCAAGGAAATTACCCCCTTGGTGATACGT TTAGCTTGGCAA GTAAACCCCGGAACGTC TTAGCTTGGCAA TGCATCTTCAGTACCACTTCTACGATATG TGCATCTTCAGT ATAAGATACATG CCGCATGGACCGGAGGGATAAGATACATG AAGCGCCATTTG CTTGCAGCAGTAGTACT AAGCGCCATTTG ATTTGCTGTTAGTTTATGTCTCGTAATAT ATTTGCTGTTAG AGTGGTTGTATG TTTTAATCTGTACGGCTAGTGGTTGTATG TCCCCATCGATC AAACGAATGAGGGTTAA TCCCCATCGATC CTACATGTCAACAGCCGCCAGGGTAGACA CTACATGTCAAC CAATGCGGTTAT AG CAATGCGGTTAT ATTCTACGTGTTATTCTACGTGTT TGTATCATCCCA TGTATCATCCCA ACGCACCCCAAT ACGCACCCCAATGCCTCTCTCATA GCCTCTCTCATA TGAATTCCGGTT TGAATTCCGGTT GTACATTTACCTGTACATTTACCT CGCCACATTTAG CGCCACATTTAG CCCAGCGTGTTG CCCAGCGTGTTGCAAGCACAGTGT CAAGCACAGTGT ATCAGAATTGTG ATCAGAATTGTG AACATGCAGATAAACATGCAGATA ACTACACCGCAT ACTACACCGCAT ATTGTCTGGGAA ATTGTCTGGGAATATCTCATATGG TATCTCATATGG AGCCTAGCTTTG AGCCTAGCTTTG GTCTAATCTTACGTCTAATCTTAC ACGACGGGGGCA ACGACGGGGGCA CCACGTTAAAGT CCACGTTAAAGTTTGTAGATACAC TTGTAGATACAC CCGAGAGTTTGT CCGAGAGTTTGT CGGGATTATACGCGGGATTATACG TTTTTGTGGTGT TTTTTGTGGTGT ATTTTAACGGGC ATTTTAACGGGCATGTTGAAGCCG ATGTTGAAGCCG TAGCATACACTG TAGCATACACTG TTGTATCCACAGTTGTATCCACAG TAGATCATTTTG TAGATCATTTTG TAAACGCAATTG TAAACGCAATTGAAGAGCGTGGAT AAGAGCGTGGAT TTCCGCCAACGG TTCCGCCAACGG CCGGTCAGCCACCCGGTCAGCCAC CGGCGACTACTA CGGCGACTACTA AACCCAAGGAAA AACCCAAGGAAATTACCCCCGTAA TTACCCCCGTAA ACCCCGGAACGT ACCCCGGAACGT CACCACTTCTACCACCACTTCTAC GATATGCCGCAT GATATGCCGCAT GGACCGGAGGGC GGACCGGAGGGCTTGCAGCAGTAG TTGCAGCAGTAG TACTTTTATGTC TACTTTTATGTC TCGTAATATTTTTCGTAATATTTT TAATCTGTACGG TAATCTGTACGG CTAAACGAATGA CTAAACGAATGAGGGTTAAAGCCG GGGTTAAAGCCG CCAGGGTAGACA CCAGGGTAGACA AGTGATAATAGGAGTGATAATAGG CTGGAGCCTCGG CTGGAGCCTCGG TGGCCATGCTTC TGGCCATGCTTCTTGCCCCTTGGG TTGCCCCTTGGG CCTCCCCCCAGC CCTCCCCCCAGC CCCTCCTCCCCTCCCTCCTCCCCT TCCTGCACCCGT TCCTGCACCCGT ACCCCCGTGGTC ACCCCCGTGGTCTTTGAATAAAGT TTTGAATAAAGT CTGAGTGGGCGG CTGAGTGGGCGG C CAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAA AAAAATCTAG SEQ ID NO: 76 SEQ ID NO: 77SEQ ID NO: 78 SEQ ID NO: 79 VZV-GE- GGGAAATAAGAG MGTVNKPVVGVLMGFGIATGGGGACAGTTAATAA GGGAAATAAGAG truncated- AGAAAAGAAGAG ITGTLRITNPVRASVLRACCTGTGGTGGGGGTAT AGAAAAGAAGAG delete_ TAAGAAGAAATA YDDFHIDEDKLDTNSVYTGATGGGGTTCGGAATT TAAGAAGAAATA from_574_-_ TAAGAGCCACCAEPYYHSDHAESSWVNRG ATCACGGGAACGTTGCG TAAGAGCCACCA Y569A TGGGGACAGTTAESSRKAYDHNSPYIWPR TATAACGAATCCGGTCA TGGGGACAGTTA Variant ATAAACCTGTGGNDYDGFLENAHEHHGVY GAGCATCCGTCTTGCGA ATAAACCTGTGG 5 TGGGGGTATTGANQGRGIDSGERLMQPTQ TACGATGATTTTCACAT TGGGGGTATTGA TGGGGTTCGGAAMSAQEDLGDDTGIHVIP CGATGAAGACAAACTGG TGGGGTTCGGAA TTATCACGGGAATLNGDDRHKIVNVDQRQ ATACAAACTCCGTATAT TTATCACGGGAA CGTTGCGTATAAYGDVFKGDLNPKPQGQR GAGCCTTACTACCATTC CGTTGCGTATAA CGAATCCGGTCALIEVSVEENHPFTLRAP AGATCATGCGGAGTCTT CGAATCCGGTCA GAGCATCCGTCTIQRIYGVRYTETWSFLP CATGGGTAAATCGGGGA GAGCATCCGTCT TGCGATACGATGSLTCTGDAAPAIQHICL GAGTCTTCGCGAAAGGC TGCGATACGATG ATTTTCACATCGKHTTCFQDVVVDVDCAE GTACGATCATAACTCAC ATTTTCACATCG ATGAAGACAAACNTKEDQLAEISYRFQGK CTTATATATGGCCACGT ATGAAGACAAAC TGGATACAAACTKEADQPWIVVNTSTLFD AATGATTATGATGGATT TGGATACAAACT CCGTATATGAGCELELDPPEIEPGVLKVL TTTAGAGAACGCACACG CCGTATATGAGC CTTACTACCATTRTEKQYLGVYIWNMRGS AACACCATGGGGTGTAT CTTACTACCATT CAGATCATGCGGDGTSTYATFLVTWKGDE AATCAGGGCCGTGGTAT CAGATCATGCGG AGTCTTCATGGGKTRNPTPAVTPQPRGAE CGATAGCGGGGAACGGT AGTCTTCATGGG TAAATCGGGGAGFHMWNYHSHVFSVGDTF TAATGCAACCCACACAA TAAATCGGGGAG AGTCTTCGCGAASLAMHLQYKIHEAPFDL ATGTCTGCACAGGAGGA AGTCTTCGCGAA AGGCGTACGATCLLEWLYVPIDPTCQPMR TCTTGGGGACGATACGG AGGCGTACGATC ATAACTCACCTTLYSTCLYHPNAPQCLSH GCATCCACGTTATCCCT ATAACTCACCTT ATATATGGCCACMNSGCTFTSPHLAQRVA ACGTTAAACGGCGATGA ATATATGGCCAC GTAATGATTATGSTVYQNCEHADNYTAYC CAGACATAAGATTGTAA GTAATGATTATG ATGGATTTTTAGLGISHMEPSFGLILHDG ATGTGGACCAACGTCAA ATGGATTTTTAG AGAACGCACACGGTTLKFVDTPESLSGLY TACGGTGACGTGTTTAA AGAACGCACACG AACACCATGGGGVFVVYFNGHVEAVAYTV AGGAGATCTTAATCCAA AACACCATGGGG TGTATAATCAGGVSTVDHFVNAIEERGFP AGCCCCAAGGCCAAAGA TGTATAATCAGG GCCGTGGTATCGPTAGQPPATTKPKEITP CTCATTGAGGTGTCAGT GCCGTGGTATCG ATAGCGGGGAACVNPGTSPLLRYAAWTGG GGAAGAGAATCACCCGT ATAGCGGGGAAC GGTTAATGCAACLAAVVLLCLVIFLICTA TTACTTTACGCGCACCG GGTTAATGCAAC CCACACAAATGTKRMRVKAARVDK ATTCAGCGGATTTATGG CCACACAAATGT CTGCACAGGAGGAGTCCGGTACACCGAGA CTGCACAGGAGG ATCTTGGGGACG CTTGGAGCTTTTTGCCGATCTTGGGGACG ATACGGGCATCC TCATTAACCTGTACGGG ATACGGGCATCC ACGTTATCCCTAAGACGCAGCGCCCGCCA ACGTTATCCCTA CGTTAAACGGCG TCCAGCATATATGTTTACGTTAAACGGCG ATGACAGACATA AAGCATACAACATGCTT ATGACAGACATA AGATTGTAAATGTCAAGACGTGGTGGTGG AGATTGTAAATG TGGACCAACGTC ATGTGGATTGCGCGGAGTGGACCAACGTC AATACGGTGACG AATACTAAAGAGGATCA AATACGGTGACG TGTTTAAAGGAGGTTGGCCGAAATCAGTT TGTTTAAAGGAG ATCTTAATCCAA ACCGTTTTCAAGGTAAGATCTTAATCCAA AGCCCCAAGGCC AAGGAAGCGGACCAACC AGCCCCAAGGCC AAAGACTCATTGGTGGATTGTTGTAAACA AAAGACTCATTG AGGTGTCAGTGG CGAGCACACTGTTTGATAGGTGTCAGTGG AAGAGAATCACC GAACTCGAATTAGACCC AAGAGAATCACC CGTTTACTTTACACCCGAGATTGAACCGG CGTTTACTTTAC GCGCACCGATTC GTGTCTTGAAAGTACTTGCGCACCGATTC AGCGGATTTATG CGGACAGAGAAACAATA AGCGGATTTATG GAGTCCGGTACACTTGGGTGTGTACATTT GAGTCCGGTACA CCGAGACTTGGA GGAACATGCGCGGCTCCCCGAGACTTGGA GCTTTTTGCCGT GATGGTACGTCTACCTA GCTTTTTGCCGT CATTAACCTGTACGCCACGTTTTTGGTCA CATTAACCTGTA CGGGAGACGCAG CCTGGAAAGGGGATGAGCGGGAGACGCAG CGCCCGCCATCC AAGACAAGAAACCCTAC CGCCCGCCATCC AGCATATATGTTGCCCGCAGTAACTCCTC AGCATATATGTT TAAAGCATACAA AACCAAGAGGGGCTGAGTAAAGCATACAA CATGCTTTCAAG TTTCATATGTGGAATTA CATGCTTTCAAG ACGTGGTGGTGGCCACTCGCATGTATTTT ACGTGGTGGTGG ATGTGGATTGCG CAGTTGGTGATACGTTTATGTGGATTGCG CGGAGAATACTA AGCTTGGCAATGCATCT CGGAGAATACTA AAGAGGATCAGTTCAGTATAAGATACATG AAGAGGATCAGT TGGCCGAAATCA AAGCGCCATTTGATTTGTGGCCGAAATCA GTTACCGTTTTC CTGTTAGAGTGGTTGTA GTTACCGTTTTC AAGGTAAGAAGGTGTCCCCATCGATCCTA AAGGTAAGAAGG AAGCGGACCAAC CATGTCAACCAATGCGGAAGCGGACCAAC CGTGGATTGTTG TTATATTCTACGTGTTT CGTGGATTGTTG TAAACACGAGCAGTATCATCCCAACGCAC TAAACACGAGCA CACTGTTTGATG CCCAATGCCTCTCTCATCACTGTTTGATG AACTCGAATTAG ATGAATTCCGGTTGTAC AACTCGAATTAG ACCCACCCGAGAATTTACCTCGCCACATT ACCCACCCGAGA TTGAACCGGGTG TAGCCCAGCGTGTTGCATTGAACCGGGTG TCTTGAAAGTAC AGCACAGTGTATCAGAA TCTTGAAAGTAC TTCGGACAGAGATTGTGAACATGCAGATA TTCGGACAGAGA AACAATACTTGG ACTACACCGCATATTGTAACAATACTTGG GTGTGTACATTT CTGGGAATATCTCATAT GTGTGTACATTT GGAACATGCGCGGGAGCCTAGCTTTGGTC GGAACATGCGCG GCTCCGATGGTA TAATCTTACACGACGGGGCTCCGATGGTA CGTCTACCTACG GGCACCACGTTAAAGTT CGTCTACCTACG CCACGTTTTTGGTGTAGATACACCCGAGA CCACGTTTTTGG TCACCTGGAAAG GTTTGTCGGGATTATACTCACCTGGAAAG GGGATGAGAAGA GTTTTTGTGGTGTATTT GGGATGAGAAGA CAAGAAACCCTATAACGGGCATGTTGAAG CAAGAAACCCTA CGCCCGCAGTAA CCGTAGCATACACTGTTCGCCCGCAGTAA CTCCTCAACCAA GTATCCACAGTAGATCA CTCCTCAACCAA GAGGGGCTGAGTTTTTGTAAACGCAATTG GAGGGGCTGAGT TTCATATGTGGA AAGAGCGTGGATTTCCGTTCATATGTGGA ATTACCACTCGC CCAACGGCCGGTCAGCC ATTACCACTCGC ATGTATTTTCAGACCGGCGACTACTAAAC ATGTATTTTCAG TTGGTGATACGT CCAAGGAAATTACCCCCTTGGTGATACGT TTAGCTTGGCAA GTAAACCCCGGAACGTC TTAGCTTGGCAA TGCATCTTCAGTACCACTTCTACGATATG TGCATCTTCAGT ATAAGATACATG CCGCATGGACCGGAGGGATAAGATACATG AAGCGCCATTTG CTTGCAGCAGTAGTACT AAGCGCCATTTG ATTTGCTGTTAGTTTATGTCTCGTAATAT ATTTGCTGTTAG AGTGGTTGTATG TTTTAATCTGTACGGCTAGTGGTTGTATG TCCCCATCGATC AAACGAATGAGGGTTAA TCCCCATCGATC CTACATGTCAACAGCCGCCAGGGTAGACA CTACATGTCAAC CAATGCGGTTAT AG CAATGCGGTTAT ATTCTACGTGTTATTCTACGTGTT TGTATCATCCCA TGTATCATCCCA ACGCACCCCAAT ACGCACCCCAATGCCTCTCTCATA GCCTCTCTCATA TGAATTCCGGTT TGAATTCCGGTT GTACATTTACCTGTACATTTACCT CGCCACATTTAG CGCCACATTTAG CCCAGCGTGTTG CCCAGCGTGTTGCAAGCACAGTGT CAAGCACAGTGT ATCAGAATTGTG ATCAGAATTGTG AACATGCAGATAAACATGCAGATA ACTACACCGCAT ACTACACCGCAT ATTGTCTGGGAA ATTGTCTGGGAATATCTCATATGG TATCTCATATGG AGCCTAGCTTTG AGCCTAGCTTTG GTCTAATCTTACGTCTAATCTTAC ACGACGGGGGCA ACGACGGGGGCA CCACGTTAAAGT CCACGTTAAAGTTTGTAGATACAC TTGTAGATACAC CCGAGAGTTTGT CCGAGAGTTTGT CGGGATTATACGCGGGATTATACG TTTTTGTGGTGT TTTTTGTGGTGT ATTTTAACGGGC ATTTTAACGGGCATGTTGAAGCCG ATGTTGAAGCCG TAGCATACACTG TAGCATACACTG TTGTATCCACAGTTGTATCCACAG TAGATCATTTTG TAGATCATTTTG TAAACGCAATTG TAAACGCAATTGAAGAGCGTGGAT AAGAGCGTGGAT TTCCGCCAACGG TTCCGCCAACGG CCGGTCAGCCACCCGGTCAGCCAC CGGCGACTACTA CGGCGACTACTA AACCCAAGGAAA AACCCAAGGAAATTACCCCCGTAA TTACCCCCGTAA ACCCCGGAACGT ACCCCGGAACGT CACCACTTCTACCACCACTTCTAC GATATGCCGCAT GATATGCCGCAT GGACCGGAGGGC GGACCGGAGGGCTTGCAGCAGTAG TTGCAGCAGTAG TACTTTTATGTC TACTTTTATGTC TCGTAATATTTTTCGTAATATTTT TAATCTGTACGG TAATCTGTACGG CTAAACGAATGA CTAAACGAATGAGGGTTAAAGCCG GGGTTAAAGCCG CCAGGGTAGACA CCAGGGTAGACA AGTGATAATAGGAGTGATAATAGG CTGGAGCCTCGG CTGGAGCCTCGG TGGCCATGCTTC TGGCCATGCTTCTTGCCCCTTGGG TTGCCCCTTGGG CCTCCCCCCAGC CCTCCCCCCAGC CCCTCCTCCCCTCCCTCCTCCCCT TCCTGCACCCGT TCCTGCACCCGT ACCCCCGTGGTC ACCCCCGTGGTCTTTGAATAAAGT TTTGAATAAAGT CTGAGTGGGCGG CTGAGTGGGCGG C CAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAA AAAAATCTAG SEQ ID NO: 80 SEQ ID NO: 81SEQ ID NO: 82 SEQ ID NO: 83 VZV-GE- GGGAAATAAGAG MGTVNKPVVGVLMGFGIATGGGGACAGTTAATAA GGGAAATAAGAG truncated- AGAAAAGAAGAG ITGTLRITNPVRASVLRACCTGTGGTGGGGGTAT AGAAAAGAAGAG delete_ TAAGAAGAAATA YDDFHIDEDKLDTNSVYTGATGGGGTTCGGAATT TAAGAAGAAATA from_574_-_ TAAGAGCCACCAEPYYHSDHAESSWVNRG ATCACGGGAACGTTGCG TAAGAGCCACCA Y569A TGGGGACAGTTAESSRKAYDHNSPYIWPR TATAACGAATCCGGTCA TGGGGACAGTTA Variant ATAAACCTGTGGNDYDGFLENAHEHHGVY GAGCATCCGTCTTGCGA ATAAACCTGTGG 6 TGGGGGTATTGANQGRGIDSGERLMQPTQ TACGATGATTTTCACAT TGGGGGTATTGA TGGGGTTCGGAAMSAQEDLGDDTGIHVIP CGATGAAGACAAACTGG TGGGGTTCGGAA TTATCACGGGAATLNGDDRHKIVNVDQRQ ATACAAACTCCGTATAT TTATCACGGGAA CGTTGCGTATAAYGDVFKGDLNPKPQGQR GAGCCTTACTACCATTC CGTTGCGTATAA CGAATCCGGTCALIEVSVEENHPFTLRAP AGATCATGCGGAGTCTT CGAATCCGGTCA GAGCATCCGTCTIQRIYGVRYTETWSFLP CATGGGTAAATCGGGGA GAGCATCCGTCT TGCGATACGATGSLTCTGDAAPAIQHICL GAGTCTTCGCGAAAAGC TGCGATACGATG ATTTTCACATCGKHTTCFQDVVVDVDCAE GTACGATCATAACTCAC ATTTTCACATCG ATGAAGACAAACNTKEDQLAEISYRFQGK CTTATATATGGCCACGT ATGAAGACAAAC TGGATACAAACTKEADQPWIVVNTSTLFD AATGATTATGATGGATT TGGATACAAACT CCGTATATGAGCELELDPPEIEPGVLKVL TTTAGAGAACGCACACG CCGTATATGAGC CTTACTACCATTRTEKQYLGVYIWNMRGS AACACCATGGGGTGTAT CTTACTACCATT CAGATCATGCGGDGTSTYATFLVTWKGDE AATCAGGGCCGTGGTAT CAGATCATGCGG AGTCTTCATGGGKTRNPTPAVTPQPRGAE CGATAGCGGGGAACGGT AGTCTTCATGGG TAAATCGGGGAGFHMWNYHSHVFSVGDTF TAATGCAACCCACACAA TAAATCGGGGAG AGTCTTCGCGAASLAMHLQYKIHEAPFDL ATGTCTGCACAGGAGGA AGTCTTCGCGAA AAGCGTACGATCLLEWLYVPIDPTCQPMR TCTTGGGGACGATACGG AAGCGTACGATC ATAACTCACCTTLYSTCLYHPNAPQCLSH GCATCCACGTTATCCCT ATAACTCACCTT ATATATGGCCACMNSGCTFTSPHLAQRVA ACGTTAAACGGCGATGA ATATATGGCCAC GTAATGATTATGSTVYQNCEHADNYTAYC CAGACATAAAATTGTAA GTAATGATTATG ATGGATTTTTAGLGISHMEPSFGLILHDG ATGTGGACCAACGTCAA ATGGATTTTTAG AGAACGCACACGGTTLKFVDTPESLSGLY TACGGTGACGTGTTTAA AGAACGCACACG AACACCATGGGGVFVVYFNGHVEAVAYTV AGGAGATCTTAATCCAA AACACCATGGGG TGTATAATCAGGVSTVDHFVNAIEERGFP AACCCCAAGGCCAAAGA TGTATAATCAGG GCCGTGGTATCGPTAGQPPATTKPKEITP CTCATTGAGGTGTCAGT GCCGTGGTATCG ATAGCGGGGAACVNPGTSPLLRYAAWTGG GGAAGAAAATCACCCGT ATAGCGGGGAAC GGTTAATGCAACLAAVVLLCLVIFLICTA TTACTTTACGCGCACCG GGTTAATGCAAC CCACACAAATGTKRMRVKAARVDK ATTCAGCGGATTTATGG CCACACAAATGT CTGCACAGGAGGAGTCCGGTACACCGAGA CTGCACAGGAGG ATCTTGGGGACG CTTGGAGCTTTTTGCCGATCTTGGGGACG ATACGGGCATCC TCATTAACCTGTACGGG ATACGGGCATCC ACGTTATCCCTAAGACGCAGCGCCCGCCA ACGTTATCCCTA CGTTAAACGGCG TCCAGCATATATGTTTACGTTAAACGGCG ATGACAGACATA AAGCATACAACATGCTT ATGACAGACATA AAATTGTAAATGTCAAGACGTGGTGGTGG AAATTGTAAATG TGGACCAACGTC ATGTGGATTGCGCGGAATGGACCAACGTC AATACGGTGACG AATACTAAAGAGGATCA AATACGGTGACG TGTTTAAAGGAGGTTGGCCGAAATCAGTT TGTTTAAAGGAG ATCTTAATCCAA ACCGTTTTCAAGGTAAGATCTTAATCCAA AACCCCAAGGCC AAGGAAGCGGACCAACC AACCCCAAGGCC AAAGACTCATTGGTGGATTGTTGTAAACA AAAGACTCATTG AGGTGTCAGTGG CGAGCACACTGTTTGATAGGTGTCAGTGG AAGAAAATCACC GAACTCGAATTAGACCC AAGAAAATCACC CGTTTACTTTACCCCCGAGATTGAACCGG CGTTTACTTTAC GCGCACCGATTC GTGTCTTGAAAGTACTTGCGCACCGATTC AGCGGATTTATG CGGACAGAGAAACAATA AGCGGATTTATG GAGTCCGGTACACTTGGGTGTGTACATTT GAGTCCGGTACA CCGAGACTTGGA GGAACATGCGCGGCTCCCCGAGACTTGGA GCTTTTTGCCGT GATGGTACGTCTACCTA GCTTTTTGCCGT CATTAACCTGTACGCCACGTTTTTGGTCA CATTAACCTGTA CGGGAGACGCAG CCTGGAAAGGGGATGAGCGGGAGACGCAG CGCCCGCCATCC AAGACAAGAAACCCTAC CGCCCGCCATCC AGCATATATGTTGCCCGCAGTAACTCCTC AGCATATATGTT TAAAGCATACAA AACCAAGAGGGGCTGAGTAAAGCATACAA CATGCTTTCAAG TTTCATATGTGGAATTA CATGCTTTCAAG ACGTGGTGGTGGCCACTCGCATGTATTTT ACGTGGTGGTGG ATGTGGATTGCG CAGTTGGTGATACGTTTATGTGGATTGCG CGGAAAATACTA AGCTTGGCAATGCATCT CGGAAAATACTA AAGAGGATCAGTTCAGTATAAGATACATG AAGAGGATCAGT TGGCCGAAATCA AAGCGCCATTTGATTTGTGGCCGAAATCA GTTACCGTTTTC CTGTTAGAGTGGTTGTA GTTACCGTTTTC AAGGTAAGAAGGTGTCCCCATCGATCCTA AAGGTAAGAAGG AAGCGGACCAAC CATGTCAACCAATGCGGAAGCGGACCAAC CGTGGATTGTTG TTATATTCTACGTGTTT CGTGGATTGTTG TAAACACGAGCAGTATCATCCCAACGCAC TAAACACGAGCA CACTGTTTGATG CCCAATGCCTCTCTCATCACTGTTTGATG AACTCGAATTAG ATGAATTCCGGTTGTAC AACTCGAATTAG ACCCCCCCGAGAATTTACCTCGCCACATT ACCCCCCCGAGA TTGAACCGGGTG TAGCCCAGCGTGTTGCATTGAACCGGGTG TCTTGAAAGTAC AGCACAGTGTATCAGAA TCTTGAAAGTAC TTCGGACAGAGATTGTGAACATGCAGATA TTCGGACAGAGA AACAATACTTGG ACTACACCGCATATTGTAACAATACTTGG GTGTGTACATTT CTGGGAATATCTCATAT GTGTGTACATTT GGAACATGCGCGGGAGCCTAGCTTTGGTC GGAACATGCGCG GCTCCGATGGTA TAATCTTACACGACGGGGCTCCGATGGTA CGTCTACCTACG GGCACCACGTTAAAGTT CGTCTACCTACG CCACGTTTTTGGTGTAGATACACCCGAGA CCACGTTTTTGG TCACCTGGAAAG GTTTGTCGGGATTATACTCACCTGGAAAG GGGATGAGAAGA GTTTTTGTGGTGTATTT GGGATGAGAAGA CAAGAAACCCTATAACGGGCATGTTGAAG CAAGAAACCCTA CGCCCGCAGTAA CCGTAGCATACACTGTTCGCCCGCAGTAA CTCCTCAACCAA GTATCCACAGTAGATCA CTCCTCAACCAA GAGGGGCTGAGTTTTTGTAAACGCAATTG GAGGGGCTGAGT TTCATATGTGGA AAGAGCGTGGATTTCCGTTCATATGTGGA ATTACCACTCGC CCAACGGCCGGTCAGCC ATTACCACTCGC ATGTATTTTCAGACCGGCGACTACTAAAC ATGTATTTTCAG TTGGTGATACGT CCAAGGAAATTACCCCCTTGGTGATACGT TTAGCTTGGCAA GTAAACCCCGGAACGTC TTAGCTTGGCAA TGCATCTTCAGTACCACTTCTACGATATG TGCATCTTCAGT ATAAGATACATG CCGCATGGACCGGAGGGATAAGATACATG AAGCGCCATTTG CTTGCAGCAGTAGTACT AAGCGCCATTTG ATTTGCTGTTAGTTTATGTCTCGTAATAT ATTTGCTGTTAG AGTGGTTGTATG TTTTAATCTGTACGGCTAGTGGTTGTATG TCCCCATCGATC AAACGAATGAGGGTTAA TCCCCATCGATC CTACATGTCAACAGCCGCCAGGGTAGACA CTACATGTCAAC CAATGCGGTTAT AG CAATGCGGTTAT ATTCTACGTGTTATTCTACGTGTT TGTATCATCCCA TGTATCATCCCA ACGCACCCCAAT ACGCACCCCAATGCCTCTCTCATA GCCTCTCTCATA TGAATTCCGGTT TGAATTCCGGTT GTACATTTACCTGTACATTTACCT CGCCACATTTAG CGCCACATTTAG CCCAGCGTGTTG CCCAGCGTGTTGCAAGCACAGTGT CAAGCACAGTGT ATCAGAATTGTG ATCAGAATTGTG AACATGCAGATAAACATGCAGATA ACTACACCGCAT ACTACACCGCAT ATTGTCTGGGAA ATTGTCTGGGAATATCTCATATGG TATCTCATATGG AGCCTAGCTTTG AGCCTAGCTTTG GTCTAATCTTACGTCTAATCTTAC ACGACGGGGGCA ACGACGGGGGCA CCACGTTAAAGT CCACGTTAAAGTTTGTAGATACAC TTGTAGATACAC CCGAGAGTTTGT CCGAGAGTTTGT CGGGATTATACGCGGGATTATACG TTTTTGTGGTGT TTTTTGTGGTGT ATTTTAACGGGC ATTTTAACGGGCATGTTGAAGCCG ATGTTGAAGCCG TAGCATACACTG TAGCATACACTG TTGTATCCACAGTTGTATCCACAG TAGATCATTTTG TAGATCATTTTG TAAACGCAATTG TAAACGCAATTGAAGAGCGTGGAT AAGAGCGTGGAT TTCCGCCAACGG TTCCGCCAACGG CCGGTCAGCCACCCGGTCAGCCAC CGGCGACTACTA CGGCGACTACTA AACCCAAGGAAA AACCCAAGGAAATTACCCCCGTAA TTACCCCCGTAA ACCCCGGAACGT ACCCCGGAACGT CACCACTTCTACCACCACTTCTAC GATATGCCGCAT GATATGCCGCAT GGACCGGAGGGC GGACCGGAGGGCTTGCAGCAGTAG TTGCAGCAGTAG TACTTTTATGTC TACTTTTATGTC TCGTAATATTTTTCGTAATATTTT TAATCTGTACGG TAATCTGTACGG CTAAACGAATGA CTAAACGAATGAGGGTTAAAGCCG GGGTTAAAGCCG CCAGGGTAGACA CCAGGGTAGACA AGTGATAATAGGAGTGATAATAGG CTGGAGCCTCGG CTGGAGCCTCGG TGGCCATGCTTC TGGCCATGCTTCTTGCCCCTTGGG TTGCCCCTTGGG CCTCCCCCCAGC CCTCCCCCCAGC CCCTCCTCCCCTCCCTCCTCCCCT TCCTGCACCCGT TCCTGCACCCGT ACCCCCGTGGTC ACCCCCGTGGTCTTTGAATAAAGT TTTGAATAAAGT CTGAGTGGGCGG CTGAGTGGGCGG C CAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAA AAAAATCTAG SEQ ID NO: 84 SEQ ID NO: 85SEQ ID NO: 86 SEQ ID NO: 87 VZV-GE- GGGAAATAAGAG MGTVNKPVVGVLMGFGIATGGGGACAGTTAATAA GGGAAATAAGAG truncated- AGAAAAGAAGAG ITGTLRITNPVRASVLRACCTGTGGTGGGGGTAT AGAAAAGAAGAG delete_ TAAGAAGAAATA YDDFHIDEDKLDTNSVYTGATGGGGTTCGGAATT TAAGAAGAAATA from_574_-_ TAAGAGCCACCAEPYYHSDHAESSWVNRG ATCACGGGAACGTTGCG TAAGAGCCACCA Y569A TGGGGACAGTTAESSRKAYDHNSPYIWPR TATAACGAATCCGGTCA TGGGGACAGTTA 7 ATAAACCTGTGGNDYDGFLENAHEHHGVY GAGCATCCGTCTTGCGA ATAAACCTGTGG TGGGGGTATTGANQGRGIDSGERLMQPTQ TACGATGATTTTCACAT TGGGGGTATTGA TGGGGTTCGGAAMSAQEDLGDDTGIHVIP CGATGAAGACAAACTGG TGGGGTTCGGAA TTATCACGGGAATLNGDDRHKIVNVDQRQ ATACAAACTCCGTATAT TTATCACGGGAA CGTTGCGTATAAYGDVFKGDLNPKPQGQR GAGCCTTACTACCATTC CGTTGCGTATAA CGAATCCGGTCALIEVSVEENHPFTLRAP AGATCATGCGGAGTCTT CGAATCCGGTCA GAGCATCCGTCTIQRIYGVRYTETWSFLP CATGGGTAAATCGGGGA GAGCATCCGTCT TGCGATACGATGSLTCTGDAAPAIQHICL GAGTCTTCGCGAAAAGC TGCGATACGATG ATTTTCACATCGKHTTCFQDVVVDVDCAE GTACGATCATAACTCAC ATTTTCACATCG ATGAAGACAAACNTKEDQLAEISYRFQGK CTTATATATGGCCACGT ATGAAGACAAAC TGGATACAAACTKEADQPWIVVNTSTLFD AATGATTATGATGGATT TGGATACAAACT CCGTATATGAGCELELDPPEIEPGVLKVL TTTAGAGAACGCACACG CCGTATATGAGC CTTACTACCATTRTEKQYLGVYIWNMRGS AACACCATGGGGTGTAT CTTACTACCATT CAGATCATGCGGDGTSTYATFLVTWKGDE AATCAGGGCCGTGGTAT CAGATCATGCGG AGTCTTCATGGGKTRNPTPAVTPQPRGAE CGATAGCGGGGAACGGT AGTCTTCATGGG TAAATCGGGGAGFHMWNYHSHVFSVGDTF TAATGCAACCCACACAA TAAATCGGGGAG AGTCTTCGCGAASLAMHLQYKIHEAPFDL ATGTCTGCACAGGAGGA AGTCTTCGCGAA AAGCGTACGATCLLEWLYVPIDPTCQPMR TCTTGGGGACGATACGG AAGCGTACGATC ATAACTCACCTTLYSTCLYHPNAPQCLSH GCATCCACGTTATCCCT ATAACTCACCTT ATATATGGCCACMNSGCTFTSPHLAQRVA ACGTTAAACGGCGATGA ATATATGGCCAC GTAATGATTATGSTVYQNCEHADNYTAYC CAGACATAAAATTGTAA GTAATGATTATG ATGGATTTTTAGLGISHMEPSFGLILHDG ATGTGGACCAACGTCAA ATGGATTTTTAG AGAACGCACACGGTTLKFVDTPESLSGLY TACGGTGACGTGTTTAA AGAACGCACACG AACACCATGGGGVFVVYFNGHVEAVAYTV AGGAGATCTTAATCCAA AACACCATGGGG TGTATAATCAGGVSTVDHFVNAIEERGFP AACCCCAAGGCCAAAGA TGTATAATCAGG GCCGTGGTATCGPTAGQPPATTKPKEITP CTCATTGAGGTGTCAGT GCCGTGGTATCG ATAGCGGGGAACVNPGTSPLLRYAAWTGG GGAAGAAAATCACCCGT ATAGCGGGGAAC GGTTAATGCAACLAAVVLLCLVIFLICTA TTACTTTACGCGCACCG GGTTAATGCAAC CCACACAAATGTKRMRVKAARVDK ATTCAGCGGATTTATGG CCACACAAATGT CTGCACAGGAGGAGTCCGGTACACCGAGA CTGCACAGGAGG ATCTTGGGGACG CTTGGAGCTTTTTGCCGATCTTGGGGACG ATACGGGCATCC TCATTAACCTGTACGGG ATACGGGCATCC ACGTTATCCCTAAGACGCAGCGCCCGCCA ACGTTATCCCTA CGTTAAACGGCG TCCAGCATATATGTTTACGTTAAACGGCG ATGACAGACATA AAGCATACAACATGCTT ATGACAGACATA AAATTGTAAATGTCAAGACGTGGTGGTGG AAATTGTAAATG TGGACCAACGTC ATGTGGATTGCGCGGAATGGACCAACGTC AATACGGTGACG AATACTAAAGAGGATCA AATACGGTGACG TGTTTAAAGGAGGTTGGCCGAAATCAGTT TGTTTAAAGGAG ATCTTAATCCAA ACCGTTTTCAAGGTAAGATCTTAATCCAA AACCCCAAGGCC AAGGAAGCGGACCAACC AACCCCAAGGCC AAAGACTCATTGGTGGATTGTTGTAAACA AAAGACTCATTG AGGTGTCAGTGG CGAGCACACTGTTTGATAGGTGTCAGTGG AAGAAAATCACC GAACTCGAATTAGACCC AAGAAAATCACC CGTTTACTTTACACCCGAGATTGAACCGG CGTTTACTTTAC GCGCACCGATTC GTGTCTTGAAAGTACTTGCGCACCGATTC AGCGGATTTATG CGGACAGAGAAACAATA AGCGGATTTATG GAGTCCGGTACACTTGGGTGTGTACATTT GAGTCCGGTACA CCGAGACTTGGA GGAACATGCGCGGCTCCCCGAGACTTGGA GCTTTTTGCCGT GATGGTACGTCTACCTA GCTTTTTGCCGT CATTAACCTGTACGCCACGTTTTTGGTCA CATTAACCTGTA CGGGAGACGCAG CCTGGAAAGGGGATGAGCGGGAGACGCAG CGCCCGCCATCC AAGACAAGAAACCCTAC CGCCCGCCATCC AGCATATATGTTGCCCGCAGTAACTCCTC AGCATATATGTT TAAAGCATACAA AACCAAGAGGGGCTGAGTAAAGCATACAA CATGCTTTCAAG TTTCATATGTGGAATTA CATGCTTTCAAG ACGTGGTGGTGGCCACTCGCATGTATTTT ACGTGGTGGTGG ATGTGGATTGCG CAGTTGGTGATACGTTTATGTGGATTGCG CGGAAAATACTA AGCTTGGCAATGCATCT CGGAAAATACTA AAGAGGATCAGTTCAGTATAAGATACATG AAGAGGATCAGT TGGCCGAAATCA AAGCGCCATTTGATTTGTGGCCGAAATCA GTTACCGTTTTC CTGTTAGAGTGGTTGTA GTTACCGTTTTC AAGGTAAGAAGGTGTCCCCATCGATCCTA AAGGTAAGAAGG AAGCGGACCAAC CATGTCAACCAATGCGGAAGCGGACCAAC CGTGGATTGTTG TTATATTCTACGTGTTT CGTGGATTGTTG TAAACACGAGCAGTATCATCCCAACGCAC TAAACACGAGCA CACTGTTTGATG CCCAATGCCTCTCTCATCACTGTTTGATG AACTCGAATTAG ATGAATTCCGGTTGTAC AACTCGAATTAG ACCCACCCGAGAATTTACCTCGCCACATT ACCCACCCGAGA TTGAACCGGGTG TAGCCCAGCGTGTTGCATTGAACCGGGTG TCTTGAAAGTAC AGCACAGTGTATCAGAA TCTTGAAAGTAC TTCGGACAGAGATTGTGAACATGCAGATA TTCGGACAGAGA AACAATACTTGG ACTACACCGCATATTGTAACAATACTTGG GTGTGTACATTT CTGGGAATATCTCATAT GTGTGTACATTT GGAACATGCGCGGGAGCCTAGCTTTGGTC GGAACATGCGCG GCTCCGATGGTA TAATCTTACACGACGGGGCTCCGATGGTA CGTCTACCTACG GGCACCACGTTAAAGTT CGTCTACCTACG CCACGTTTTTGGTGTAGATACACCCGAGA CCACGTTTTTGG TCACCTGGAAAG GTTTGTCGGGATTATACTCACCTGGAAAG GGGATGAGAAGA GTTTTTGTGGTGTATTT GGGATGAGAAGA CAAGAAACCCTATAACGGGCATGTTGAAG CAAGAAACCCTA CGCCCGCAGTAA CCGTAGCATACACTGTTCGCCCGCAGTAA CTCCTCAACCAA GTATCCACAGTAGATCA CTCCTCAACCAA GAGGGGCTGAGTTTTTGTAAACGCAATTG GAGGGGCTGAGT TTCATATGTGGA AAGAGCGTGGATTTCCGTTCATATGTGGA ATTACCACTCGC CCAACGGCCGGTCAGCC ATTACCACTCGC ATGTATTTTCAGACCGGCGACTACTAAAC ATGTATTTTCAG TTGGTGATACGT CCAAGGAAATTACCCCCTTGGTGATACGT TTAGCTTGGCAA GTAAACCCCGGAACGTC TTAGCTTGGCAA TGCATCTTCAGTACCACTTCTACGATATG TGCATCTTCAGT ATAAGATACATG CCGCATGGACCGGAGGGATAAGATACATG AAGCGCCATTTG CTTGCAGCAGTAGTACT AAGCGCCATTTG ATTTGCTGTTAGTTTATGTCTCGTAATAT ATTTGCTGTTAG AGTGGTTGTATG TTTTAATCTGTACGGCTAGTGGTTGTATG TCCCCATCGATC AAACGAATGAGGGTTAA TCCCCATCGATC CTACATGTCAACAGCCGCCAGGGTAGACA CTACATGTCAAC CAATGCGGTTAT AG CAATGCGGTTAT ATTCTACGTGTTATTCTACGTGTT TGTATCATCCCA TGTATCATCCCA ACGCACCCCAAT ACGCACCCCAATGCCTCTCTCATA GCCTCTCTCATA TGAATTCCGGTT TGAATTCCGGTT GTACATTTACCTGTACATTTACCT CGCCACATTTAG CGCCACATTTAG CCCAGCGTGTTG CCCAGCGTGTTGCAAGCACAGTGT CAAGCACAGTGT ATCAGAATTGTG ATCAGAATTGTG AACATGCAGATAAACATGCAGATA ACTACACCGCAT ACTACACCGCAT ATTGTCTGGGAA ATTGTCTGGGAATATCTCATATGG TATCTCATATGG AGCCTAGCTTTG AGCCTAGCTTTG GTCTAATCTTACGTCTAATCTTAC ACGACGGGGGCA ACGACGGGGGCA CCACGTTAAAGT CCACGTTAAAGTTTGTAGATACAC TTGTAGATACAC CCGAGAGTTTGT CCGAGAGTTTGT CGGGATTATACGCGGGATTATACG TTTTTGTGGTGT TTTTTGTGGTGT ATTTTAACGGGC ATTTTAACGGGCATGTTGAAGCCG ATGTTGAAGCCG TAGCATACACTG TAGCATACACTG TTGTATCCACAGTTGTATCCACAG TAGATCATTTTG TAGATCATTTTG TAAACGCAATTG TAAACGCAATTGAAGAGCGTGGAT AAGAGCGTGGAT TTCCGCCAACGG TTCCGCCAACGG CCGGTCAGCCACCCGGTCAGCCAC CGGCGACTACTA CGGCGACTACTA AACCCAAGGAAA AACCCAAGGAAATTACCCCCGTAA TTACCCCCGTAA ACCCCGGAACGT ACCCCGGAACGT CACCACTTCTACCACCACTTCTAC GATATGCCGCAT GATATGCCGCAT GGACCGGAGGGC GGACCGGAGGGCTTGCAGCAGTAG TTGCAGCAGTAG TACTTTTATGTC TACTTTTATGTC TCGTAATATTTTTCGTAATATTTT TAATCTGTACGG TAATCTGTACGG CTAAACGAATGA CTAAACGAATGAGGGTTAAAGCCG GGGTTAAAGCCG CCAGGGTAGACA CCAGGGTAGACA AGTGATAATAGGAGTGATAATAGG CTGGAGCCTCGG CTGGAGCCTCGG TGGCCATGCTTC TGGCCATGCTTCTTGCCCCTTGGG TTGCCCCTTGGG CCTCCCCCCAGC CCTCCCCCCAGC CCCTCCTCCCCTCCCTCCTCCCCT TCCTGCACCCGT TCCTGCACCCGT ACCCCCGTGGTC ACCCCCGTGGTCTTTGAATAAAGT TTTGAATAAAGT CTGAGTGGGCGG CTGAGTGGGCGG C CAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAA AAAAATCTAG SEQ ID NO: 88 SEQ ID NO: 89SEQ ID NO: 90 SEQ ID NO: 91 VZV-GE- GGGAAATAAGAG MGTVNKPVVGVLMGFGIATGGGGACAGTTAATAA GGGAAATAAGAG truncated- AGAAAAGAAGAG ITGTLRITNPVRASVLRACCTGTGGTGGGCGTAT AGAAAAGAAGAG delete_ TAAGAAGAAATA YDDFHIDEDKLDTNSVYTGATGGGGTTCGGAATT TAAGAAGAAATA from_574_-_ TAAGAGCCACCAEPYYHSDHAESSWVNRG ATCACGGGAACGTTGCG TAAGAGCCACCA Y569A TGGGGACAGTTAESSRKAYDHNSPYIWPR TATAACGAATCCGGTCA TGGGGACAGTTA Variant ATAAACCTGTGGNDYDGFLENAHEHHGVY GAGCATCCGTCTTGCGA ATAAACCTGTGG 8 TGGGCGTATTGANQGRGIDSGERLMQPTQ TACGATGATTTTCACAT TGGGCGTATTGA TGGGGTTCGGAAMSAQEDLGDDTGIHVIP CGATGAAGACAAACTGG TGGGGTTCGGAA TTATCACGGGAATLNGDDRHKIVNVDQRQ ATACAAACTCCGTATAT TTATCACGGGAA CGTTGCGTATAAYGDVFKGDLNPKPQGQR GAGCCTTACTACCATTC CGTTGCGTATAA CGAATCCGGTCALIEVSVEENHPFTLRAP AGATCATGCGGAGTCTT CGAATCCGGTCA GAGCATCCGTCTIQRIYGVRYTETWSFLP CATGGGTAAATCGGGGA GAGCATCCGTCT TGCGATACGATGSLTCTGDAAPAIQHICL GAGTCTTCGCGAAAGGC TGCGATACGATG ATTTTCACATCGKHTTCFQDVVVDVDCAE GTACGATCATAACTCAC ATTTTCACATCG ATGAAGACAAACNTKEDQLAEISYRFQGK CTTATATATGGCCACGT ATGAAGACAAAC TGGATACAAACTKEADQPWIVVNTSTLFD AATGATTATGATGGATT TGGATACAAACT CCGTATATGAGCELELDPPEIEPGVLKVL CTTAGAGAACGCACACG CCGTATATGAGC CTTACTACCATTRTEKQYLGVYIWNMRGS AACACCATGGGGTGTAT CTTACTACCATT CAGATCATGCGGDGTSTYATFLVTWKGDE AATCAGGGCCGTGGTAT CAGATCATGCGG AGTCTTCATGGGKTRNPTPAVTPQPRGAE CGATAGCGGGGAACGGT AGTCTTCATGGG TAAATCGGGGAGFHMWNYHSHVFSVGDTF TAATGCAACCCACACAA TAAATCGGGGAG AGTCTTCGCGAASLAMHLQYKIHEAPFDL ATGTCTGCACAGGAGGA AGTCTTCGCGAA AGGCGTACGATCLLEWLYVPIDPTCQPMR TCTTGGGGACGATACGG AGGCGTACGATC ATAACTCACCTTLYSTCLYHPNAPQCLSH GCATCCACGTTATCCCT ATAACTCACCTT ATATATGGCCACMNSGCTFTSPHLAQRVA ACGTTAAACGGCGATGA ATATATGGCCAC GTAATGATTATGSTVYQNCEHADNYTAYC CAGACATAAGATTGTAA GTAATGATTATG ATGGATTCTTAGLGISHMEPSFGLILHDG ATGTGGACCAACGTCAA ATGGATTCTTAG AGAACGCACACGGTTLKFVDTPESLSGLY TACGGTGACGTGTTTAA AGAACGCACACG AACACCATGGGGVFVVYFNGHVEAVAYTV AGGAGATCTTAATCCAA AACACCATGGGG TGTATAATCAGGVSTVDHFVNAIEERGFP AGCCCCAAGGCCAAAGA TGTATAATCAGG GCCGTGGTATCGPTAGQPPATTKPKEITP CTCATTGAGGTGTCAGT GCCGTGGTATCG ATAGCGGGGAACVNPGTSPLLRYAAWTGG GGAAGAGAATCACCCGT ATAGCGGGGAAC GGTTAATGCAACLAAVVLLCLVIFLICTA TTACTTTACGCGCACCG GGTTAATGCAAC CCACACAAATGTKRMRVKAARVDK ATTCAGCGGATTTATGG CCACACAAATGT CTGCACAGGAGGAGTCCGGTACACCGAGA CTGCACAGGAGG ATCTTGGGGACG CTTGGAGCTTCTTGCCGATCTTGGGGACG ATACGGGCATCC TCATTAACCTGTACGGG ATACGGGCATCC ACGTTATCCCTAAGACGCAGCGCCCGCCA ACGTTATCCCTA CGTTAAACGGCG TCCAGCATATATGTTTACGTTAAACGGCG ATGACAGACATA AAGCATACAACATGCTT ATGACAGACATA AGATTGTAAATGTCAAGACGTGGTGGTGG AGATTGTAAATG TGGACCAACGTC ATGTGGATTGCGCGGAGTGGACCAACGTC AATACGGTGACG AATACTAAAGAGGATCA AATACGGTGACG TGTTTAAAGGAGGTTGGCCGAAATCAGTT TGTTTAAAGGAG ATCTTAATCCAA ACCGTTTTCAAGGTAAGATCTTAATCCAA AGCCCCAAGGCC AAGGAAGCGGACCAACC AGCCCCAAGGCC AAAGACTCATTGGTGGATTGTTGTAAACA AAAGACTCATTG AGGTGTCAGTGG CGAGCACACTGTTTGATAGGTGTCAGTGG AAGAGAATCACC GAACTCGAATTAGACCC AAGAGAATCACC CGTTTACTTTACACCCGAGATTGAACCGG CGTTTACTTTAC GCGCACCGATTC GTGTCTTGAAAGTACTTGCGCACCGATTC AGCGGATTTATG CGGACAGAGAAACAATA AGCGGATTTATG GAGTCCGGTACACTTGGGTGTGTACATTT GAGTCCGGTACA CCGAGACTTGGA GGAACATGCGCGGCTCCCCGAGACTTGGA GCTTCTTGCCGT GATGGTACGTCTACCTA GCTTCTTGCCGT CATTAACCTGTACGCCACGTTCTTGGTCA CATTAACCTGTA CGGGAGACGCAG CCTGGAAAGGGGATGAGCGGGAGACGCAG CGCCCGCCATCC AAGACAAGAAACCCTAC CGCCCGCCATCC AGCATATATGTTGCCCGCAGTAACTCCTC AGCATATATGTT TAAAGCATACAA AACCAAGAGGGGCTGAGTAAAGCATACAA CATGCTTTCAAG TTTCATATGTGGAATTA CATGCTTTCAAG ACGTGGTGGTGGCCACTCGCATGTATTTT ACGTGGTGGTGG ATGTGGATTGCG CAGTTGGTGATACGTTTATGTGGATTGCG CGGAGAATACTA AGCTTGGCAATGCATCT CGGAGAATACTA AAGAGGATCAGTTCAGTATAAGATACATG AAGAGGATCAGT TGGCCGAAATCA AAGCGCCATTTGATTTGTGGCCGAAATCA GTTACCGTTTTC CTGTTAGAGTGGTTGTA GTTACCGTTTTC AAGGTAAGAAGGTGTCCCCATCGATCCTA AAGGTAAGAAGG AAGCGGACCAAC CATGTCAACCAATGCGGAAGCGGACCAAC CGTGGATTGTTG TTATATTCTACGTGTTT CGTGGATTGTTG TAAACACGAGCAGTATCATCCCAACGCAC TAAACACGAGCA CACTGTTTGATG CCCAATGCCTCTCTCATCACTGTTTGATG AACTCGAATTAG ATGAATTCCGGTTGTAC AACTCGAATTAG ACCCACCCGAGAATTTACCTCGCCACATT ACCCACCCGAGA TTGAACCGGGTG TAGCCCAGCGTGTTGCATTGAACCGGGTG TCTTGAAAGTAC AGCACAGTGTATCAGAA TCTTGAAAGTAC TTCGGACAGAGATTGTGAACATGCAGATA TTCGGACAGAGA AACAATACTTGG ACTACACCGCATATTGTAACAATACTTGG GTGTGTACATTT CTGGGAATATCTCATAT GTGTGTACATTT GGAACATGCGCGGGAGCCTAGCTTTGGTC GGAACATGCGCG GCTCCGATGGTA TAATCTTACACGACGGAGCTCCGATGGTA CGTCTACCTACG GGCACCACGTTAAAGTT CGTCTACCTACG CCACGTTCTTGGTGTAGATACACCCGAGA CCACGTTCTTGG TCACCTGGAAAG GTTTGTCGGGATTATACTCACCTGGAAAG GGGATGAGAAGA GTCTTTGTGGTGTATTT GGGATGAGAAGA CAAGAAACCCTATAACGGGCATGTTGAAG CAAGAAACCCTA CGCCCGCAGTAA CCGTAGCATACACTGTTCGCCCGCAGTAA CTCCTCAACCAA GTATCCACAGTAGATCA CTCCTCAACCAA GAGGGGCTGAGTTTTTGTAAACGCAATTG GAGGGGCTGAGT TTCATATGTGGA AAGAGCGTGGATTTCCGTTCATATGTGGA ATTACCACTCGC CCAACGGCCGGTCAGCC ATTACCACTCGC ATGTATTTTCAGACCGGCGACTACTAAAC ATGTATTTTCAG TTGGTGATACGT CCAAGGAAATTACGCCCTTGGTGATACGT TTAGCTTGGCAA GTAAACCCCGGAACGTC TTAGCTTGGCAA TGCATCTTCAGTACCACTTCTACGATATG TGCATCTTCAGT ATAAGATACATG CCGCATGGACCGGAGGGATAAGATACATG AAGCGCCATTTG CTTGCAGCAGTAGTACT AAGCGCCATTTG ATTTGCTGTTAGTTTATGTCTCGTAATAT ATTTGCTGTTAG AGTGGTTGTATG TCTTAATCTGTACGGCTAGTGGTTGTATG TCCCCATCGATC AAACGAATGAGGGTTAA TCCCCATCGATC CTACATGTCAACAGCCGCCAGGGTAGACA CTACATGTCAAC CAATGCGGTTAT AG CAATGCGGTTAT ATTCTACGTGTTATTCTACGTGTT TGTATCATCCCA TGTATCATCCCA ACGCACCCCAAT ACGCACCCCAATGCCTCTCTCATA GCCTCTCTCATA TGAATTCCGGTT TGAATTCCGGTT GTACATTTACCTGTACATTTACCT CGCCACATTTAG CGCCACATTTAG CCCAGCGTGTTG CCCAGCGTGTTGCAAGCACAGTGT CAAGCACAGTGT ATCAGAATTGTG ATCAGAATTGTG AACATGCAGATAAACATGCAGATA ACTACACCGCAT ACTACACCGCAT ATTGTCTGGGAA ATTGTCTGGGAATATCTCATATGG TATCTCATATGG AGCCTAGCTTTG AGCCTAGCTTTG GTCTAATCTTACGTCTAATCTTAC ACGACGGAGGCA ACGACGGAGGCA CCACGTTAAAGT CCACGTTAAAGTTTGTAGATACAC TTGTAGATACAC CCGAGAGTTTGT CCGAGAGTTTGT CGGGATTATACGCGGGATTATACG TCTTTGTGGTGT TCTTTGTGGTGT ATTTTAACGGGC ATTTTAACGGGCATGTTGAAGCCG ATGTTGAAGCCG TAGCATACACTG TAGCATACACTG TTGTATCCACAGTTGTATCCACAG TAGATCATTTTG TAGATCATTTTG TAAACGCAATTG TAAACGCAATTGAAGAGCGTGGAT AAGAGCGTGGAT TTCCGCCAACGG TTCCGCCAACGG CCGGTCAGCCACCCGGTCAGCCAC CGGCGACTACTA CGGCGACTACTA AACCCAAGGAAA AACCCAAGGAAATTACGCCCGTAA TTACGCCCGTAA ACCCCGGAACGT ACCCCGGAACGT CACCACTTCTACCACCACTTCTAC GATATGCCGCAT GATATGCCGCAT GGACCGGAGGGC GGACCGGAGGGCTTGCAGCAGTAG TTGCAGCAGTAG TACTTTTATGTC TACTTTTATGTC TCGTAATATTCTTCGTAATATTCT TAATCTGTACGG TAATCTGTACGG CTAAACGAATGA CTAAACGAATGAGGGTTAAAGCCG GGGTTAAAGCCG CCAGGGTAGACA CCAGGGTAGACA AGTGATAATAGGAGTGATAATAGG CTGGAGCCTCGG CTGGAGCCTCGG TGGCCATGCTTC TGGCCATGCTTCTTGCCCCTTGGG TTGCCCCTTGGG CCTCCCCCCAGC CCTCCCCCCAGC CCCTCCTCCCCTCCCTCCTCCCCT TCCTGCACCCGT TCCTGCACCCGT ACCCCCGTGGTC ACCCCCGTGGTCTTTGAATAAAGT TTTGAATAAAGT CTGAGTGGGCGG CTGAGTGGGCGG C CAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAA AAAAATCTAG VZV mRNA Sequences mRNA SEQ IDName(s) mRNA Sequence (assumes T100 tail) NO VZV_gE_G*GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGA 92 OkaGCCACCAUGGGGACAGUGAAUAAGCCGGUUGUGGGCGUGCUUAUGGGCUUUGGGAUUAUUACCGGUACAUUACGAAUUACCAAUCCAGUGCGCGCCAGUGUGCUGCGUUACGACGACUUUCACAUUGACGAGGAUAAGCUGGAUACUAACAGCGUGUACGAACCUUAUUACCACUCAGAUCAUGCCGAAUCAAGCUGGGUUAAUAGAGGAGAAAGCAGCCGAAAAGCCUACGACCACAACUCACCUUAUAUUUGGCCCAGAAACGAUUAUGACGGUUUCCUGGAAAACGCACAUGAACACCAUGGAGUCUACAACCAAGGCAGGGGAAUCGACAGUGGCGAGCGUCUUAUGCAGCCAACACAGAUGUCGGCACAGGAGGAUCUCGGUGAUGACACCGGCAUACACGUGAUUCCCACAUUAAACGGCGACGACAGACAUAAGAUCGUCAAUGUGGAUCAGCGUCAGUAUGGGGAUGUCUUUAAAGGCGAUUUGAAUCCAAAGCCCCAAGGACAGAGACUGAUCGAGGUCUCUGUAGAAGAAAAUCACCCCUUCACUUUGCGCGCUCCAAUCCAGAGGAUUUACGGGGUGCGUUAUACCGAAACUUGGAGUUUCUUGCCGUCACUGACGUGUACGGGGGAUGCCGCCCCCGCAAUCCAGCACAUCUGUCUGAAACACACCACAUGCUUUCAGGACGUGGUUGUGGAUGUGGAUUGCGCGGAAAACACAAAAGAAGACCAACUCGCCGAAAUCAGCUAUCGUUUUCAGGGUAAAAAAGAGGCCGACCAACCGUGGAUUGUUGUGAAUACGAGCACGCUCUUCGAUGAGCUUGAACUCGAUCCCCCGGAAAUCGAGCCUGGGGUUCUAAAAGUGUUGAGGACCGAGAAGCAGUACCUCGGGGUUUAUAUCUGGAAUAUGAGAGGCUCCGAUGGCACCUCUACCUACGCAACGUUUCUGGUUACCUGGAAGGGAGACGAGAAGACACGGAAUCCAACGCCCGCUGUGACCCCUCAGCCUAGGGGAGCCGAAUUCCACAUGUGGAACUAUCACUCCCAUGUAUUCAGUGUGGGUGACACUUUCAGCCUGGCCAUGCACCUGCAGUAUAAGAUUCACGAGGCACCCUUCGACCUCCUGCUGGAGUGGUUGUACGUACCUAUUGAUCCCACUUGUCAGCCCAUGCGCCUGUACUCCACUUGCUUGUACCACCCCAAUGCACCACAGUGUCUAUCACACAUGAACUCCGGGUGUACCUUUACUUCACCCCAUCUUGCCCAGCGGGUCGCCAGCACAGUGUAUCAGAACUGUGAGCAUGCUGACAACUAUACUGCUUAUUGCCUCGGAAUAUCCCAUAUGGAGCCAAGCUUCGGGCUCAUACUGCACGAUGGUGGUACGACACUCAAGUUCGUGGACACCCCCGAAAGCCUUUCUGGCUUGUACGUGUUCGUGGUCUACUUCAAUGGACAUGUGGAGGCAGUGGCUUACACAGUGGUUUCGACAGUUGAUCACUUUGUAAAUGCCAUUGAGGAACGCGGCUUCCCGCCUACAGCGGGCCAGCCCCCUGCGACAACAAAACCAAAAGAGAUUACGCCCGUUAAUCCUGGGACUAGUCCAUUGCUGAGGUAUGCCGCCUGGACUGGCGGUCUGGCGGCCGUGGUACUUCUGUGUUUAGUCAUAUUUCUGAUCUGUACCGCUAAACGUAUGCGGGUCAAGGCUUACCGUGUUGACAAGUCUCCUUACAAUCAGUCAAUGUACUAUGCAGGACUCCCUGUUGACGAUUUCGAAGACUCAGAGAGUACAGACACAGAAGAAGAAUUCGGAAACGCUAUAGGUGGCUCUCACGGAGGUAGCUCGUAUACAGUGUACAUCGAUAAAACCAGAUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAUCUAG VZV_gE_G*GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGA 135 full_indel_GCCACCAUGGGGACAGUUAAUAAACCUGUGGUGGGGGUAUUGA fixedUGGGGUUCGGAAUUAUCACGGGAACGUUGCGUAUAACGAAUCCGGUCAGAGCAUCCGUCUUGCGAUACGAUGAUUUUCACAUCGAUGAAGACAAACUGGAUACAAACUCCGUAUAUGAGCCUUACUACCAUUCAGAUCAUGCGGAGUCUUCAUGGGUAAAUCGGGGAGAGUCUUCGCGAAAAGCGUACGAUCAUAACUCACCUUAUAUAUGGCCACGUAAUGAUUAUGAUGGAUUUUUAGAGAACGCACACGAACACCAUGGGGUGUAUAAUCAGGGCCGUGGUAUCGAUAGCGGGGAACGGUUAAUGCAACCCACACAAAUGUCUGCACAGGAGGAUCUUGGGGACGAUACGGGCAUCCACGUUAUCCCUACGUUAAACGGCGAUGACAGACAUAAAAUUGUAAAUGUGGACCAACGUCAAUACGGUGACGUGUUUAAAGGAGAUCUUAAUCCAAAACCCCAAGGCCAAAGACUCAUUGAGGUGUCAGUGGAAGAAAAUCACCCGUUUACUUUACGCGCACCGAUUCAGCGGAUUUAUGGAGUCCGGUACACCGAGACUUGGAGCUUUUUGCCGUCAUUAACCUGUACGGGAGACGCAGCGCCCGCCAUCCAGCAUAUAUGUUUAAAGCAUACAACAUGCUUUCAAGACGUGGUGGUGGAUGUGGAUUGCGCGGAAAAUACUAAAGAGGAUCAGUUGGCCGAAAUCAGUUACCGUUUUCAAGGUAAGAAGGAAGCGGACCAACCGUGGAUUGUUGUAAACACGAGCACACUGUUUGAUGAACUCGAAUUAGACCCACCCGAGAUUGAACCGGGUGUCUUGAAAGUACUUCGGACAGAGAAACAAUACUUGGGUGUGUACAUUUGGAACAUGCGCGGCUCCGAUGGUACGUCUACCUACGCCACGUUUUUGGUCACCUGGAAAGGGGAUGAGAAGACAAGAAACCCUACGCCCGCAGUAACUCCUCAACCAAGAGGGGCUGAGUUUCAUAUGUGGAAUUACCACUCGCAUGUAUUUUCAGUUGGUGAUACGUUUAGCUUGGCAAUGCAUCUUCAGUAUAAGAUACAUGAAGCGCCAUUUGAUUUGCUGUUAGAGUGGUUGUAUGUCCCCAUCGAUCCUACAUGUCAACCAAUGCGGUUAUAUUCUACGUGUUUGUAUCAUCCCAACGCACCCCAAUGCCUCUCUCAUAUGAAUUCCGGUUGUACAUUUACCUCGCCACAUUUAGCCCAGCGUGUUGCAAGCACAGUGUAUCAGAAUUGUGAACAUGCAGAUAACUACACCGCAUAUUGUCUGGGAAUAUCUCAUAUGGAGCCUAGCUUUGGUCUAAUCUUACACGACGGGGGCACCACGUUAAAGUUUGUAGAUACACCCGAGAGUUUGUCGGGAUUAUACGUUUUUGUGGUGUAUUUUAACGGGCAUGUUGAAGCCGUAGCAUACACUGUUGUAUCCACAGUAGAUCAUUUUGUAAACGCAAUUGAAGAGCGUGGAUUUCCGCCAACGGCCGGUCAGCCACCGGCGACUACUAAACCCAAGGAAAUUACCCCCGUAAACCCCGGAACGUCACCACUUCUACGAUAUGCCGCAUGGACCGGAGGGCUUGCAGCAGUAGUACUUUUAUGUCUCGUAAUAUUUUUAAUCUGUACGGCUAAACGAAUGAGGGUUAAAGCCUACAGGGUAGACAAGUCUCCUUACAAUCAGUCAAUGUACUAUGCAGGACUCCCUGUUGACGAUUUCGAAGACUCAGAGAGUACAGACACAGAAGAAGAAUUCGGAAACGCUAUAGGUGGCUCUCACGGAGGUAGCUCGUAUACAGUGUACAUCGAUAAAACCAGAUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC VZV_gE_G*GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGA 93 Oka_hIgkappaGCCACCAUGGAGACUCCCGCUCAGCUACUGUUCCUCCUGCUCCUUUGGCUGCCUGAUACUACAGGCUCUGUUUUGCGGUACGACGACUUUCACAUCGAUGAGGACAAGCUCGACACUAAUAGCGUGUAUGAGCCCUACUACCAUUCAGAUCACGCCGAGUCCUCUUGGGUGAACAGGGGUGAAAGUUCUAGGAAAGCCUAUGAUCACAACAGCCCUUAUAUUUGGCCACGGAAUGAUUACGACGGAUUUCUCGAAAAUGCCCACGAGCAUCACGGAGUGUACAACCAGGGCCGUGGAAUCGACUCUGGGGAGAGAUUGAUGCAACCUACACAGAUGAGCGCCCAGGAAGAUCUCGGGGAUGAUACAGGAAUUCACGUUAUCCCUACAUUAAACGGAGAUGACCGCCACAAAAUCGUCAAUGUCGAUCAAAGACAGUAUGGAGAUGUGUUCAAAGGCGAUCUCAACCCUAAGCCGCAGGGCCAGAGACUCAUUGAGGUGUCUGUCGAAGAGAACCACCCUUUCACUCUGCGCGCUCCCAUUCAGAGAAUCUAUGGAGUUCGCUAUACGGAGACUUGGUCAUUCCUUCCUUCCCUGACAUGCACCGGAGACGCCGCCCCUGCCAUUCAGCACAUAUGCCUGAAACAUACCACCUGUUUCCAGGAUGUGGUGGUUGAUGUUGAUUGUGCUGAAAAUACCAAGGAAGACCAACUGGCCGAGAUUAGUUACCGGUUCCAAGGGAAAAAGGAAGCCGACCAGCCAUGGAUUGUGGUUAAUACAAGCACUCUGUUCGAUGAGCUCGAGCUGGAUCCCCCCGAGAUAGAACCCGGAGUUCUGAAAGUGCUCCGGACAGAAAAACAAUAUCUGGGAGUCUACAUAUGGAACAUGCGCGGUUCCGAUGGGACCUCCACUUAUGCAACCUUUCUCGUCACGUGGAAGGGAGAUGAGAAAACUAGGAAUCCCACACCCGCUGUCACACCACAGCCAAGAGGGGCUGAGUUCCAUAUGUGGAACUAUCAUAGUCACGUGUUUAGUGUCGGAGAUACGUUUUCAUUGGCUAUGCAUCUCCAGUACAAGAUUCAUGAGGCUCCCUUCGAUCUGUUGCUUGAGUGGUUGUACGUCCCGAUUGACCCGACCUGCCAGCCCAUGCGACUGUACAGCACCUGUCUCUACCAUCCAAACGCUCCGCAAUGUCUGAGCCACAUGAACUCUGGGUGUACUUUCACCAGUCCCCACCUCGCCCAGCGGGUGGCCUCUACUGUUUACCAGAACUGUGAGCACGCCGACAACUACACCGCAUACUGCCUCGGUAUUUCUCACAUGGAACCCUCCUUCGGACUCAUCCUGCACGAUGGGGGCACUACCCUGAAGUUCGUUGAUACGCCAGAAUCUCUGUCUGGGCUCUAUGUUUUCGUGGUCUACUUCAAUGGCCAUGUCGAGGCCGUGGCCUAUACUGUCGUUUCUACCGUGGAUCAUUUUGUGAACGCCAUCGAAGAACGGGGAUUCCCCCCUACGGCAGGCCAGCCGCCUGCAACCACCAAGCCCAAGGAAAUAACACCAGUGAACCCUGGCACCUCACCUCUCCUAAGAUAUGCCGCGUGGACAGGGGGACUGGCGGCAGUGGUGCUCCUCUGUCUCGUGAUCUUUCUGAUCUGUACAGCCAAGAGGAUGAGGGUCAAGGCUUAUAGAGUGGACAAGUCCCCCUACAAUCAGUCAAUGUACUACGCCGGCCUUCCCGUUGAUGAUUUUGAGGAUUCCGAGUCCACAGAUACUGAGGAAGAGUUCGGUAACGCUAUAGGCGGCUCUCACGGGGGUUCAAGCUACACGGUUUACAUUGACAAGACACGCUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AUCUAG VZV-GE-G*GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGA 94 delete-562GCCACCAUGGGGACAGUUAAUAAACCUGUGGUGGGGGUAUUGAUGGGGUUCGGAAUUAUCACGGGAACGUUGCGUAUAACGAAUCCGGUCAGAGCAUCCGUCUUGCGAUACGAUGAUUUUCACAUCGAUGAAGACAAACUGGAUACAAACUCCGUAUAUGAGCCUUACUACCAUUCAGAUCAUGCGGAGUCUUCAUGGGUAAAUCGGGGAGAGUCUUCGCGAAAAGCGUACGAUCAUAACUCACCUUAUAUAUGGCCACGUAAUGAUUAUGAUGGAUUUUUAGAGAACGCACACGAACACCAUGGGGUGUAUAAUCAGGGCCGUGGUAUCGAUAGCGGGGAACGGUUAAUGCAACCCACACAAAUGUCUGCACAGGAGGAUCUUGGGGACGAUACGGGCAUCCACGUUAUCCCUACGUUAAACGGCGAUGACAGACAUAAAAUUGUAAAUGUGGACCAACGUCAAUACGGUGACGUGUUUAAAGGAGAUCUUAAUCCAAAACCCCAAGGCCAAAGACUCAUUGAGGUGUCAGUGGAAGAAAAUCACCCGUUUACUUUACGCGCACCGAUUCAGCGGAUUUAUGGAGUCCGGUACACCGAGACUUGGAGCUUUUUGCCGUCAUUAACCUGUACGGGAGACGCAGCGCCCGCCAUCCAGCAUAUAUGUUUAAAACAUACAACAUGCUUUCAAGACGUGGUGGUGGAUGUGGAUUGCGCGGAAAAUACUAAAGAGGAUCAGUUGGCCGAAAUCAGUUACCGUUUUCAAGGUAAGAAGGAAGCGGACCAACCGUGGAUUGUUGUAAACACGAGCACACUGUUUGAUGAACUCGAAUUAGACCCCCCCGAGAUUGAACCGGGUGUCUUGAAAGUACUUCGGACAGAAAAACAAUACUUGGGUGUGUACAUUUGGAACAUGCGCGGCUCCGAUGGUACGUCUACCUACGCCACGUUUUUGGUCACCUGGAAAGGGGAUGAAAAAACAAGAAACCCUACGCCCGCAGUAACUCCUCAACCAAGAGGGGCUGAGUUUCAUAUGUGGAAUUACCACUCGCAUGUAUUUUCAGUUGGUGAUACGUUUAGCUUGGCAAUGCAUCUUCAGUAUAAGAUACAUGAAGCGCCAUUUGAUUUGCUGUUAGAGUGGUUGUAUGUCCCCAUCGAUCCUACAUGUCAACCAAUGCGGUUAUAUUCUACGUGUUUGUAUCAUCCCAACGCACCCCAAUGCCUCUCUCAUAUGAAUUCCGGUUGUACAUUUACCUCGCCACAUUUAGCCCAGCGUGUUGCAAGCACAGUGUAUCAAAAUUGUGAACAUGCAGAUAACUACACCGCAUAUUGUCUGGGAAUAUCUCAUAUGGAGCCUAGCUUUGGUCUAAUCUUACACGACGGGGGCACCACGUUAAAGUUUGUAGAUACACCCGAGAGUUUGUCGGGAUUAUACGUUUUUGUGGUGUAUUUUAACGGGCAUGUUGAAGCCGUAGCAUACACUGUUGUAUCCACAGUAGAUCAUUUUGUAAACGCAAUUGAAGAGCGUGGAUUUCCGCCAACGGCCGGUCAGCCACCGGCGACUACUAAACCCAAGGAAAUUACCCCCGUAAACCCCGGAACGUCACCACUUCUACGAUAUGCCGCAUGGACCGGAGGGCUUGCAGCAGUAGUACUUUUAUGUCUCGUAAUAUUUUUAAUCUGUACGGCUUGAUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAUCUA VZV-GE-G*GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGA 95 delete-GCCACCAUGGAAACCCCGGCGCAGCUGCUGUUUCUGCUGCUGCU 562-GUGGCUGCCGGAUACCACCGGCUCCGUCUUGCGAUACGAUGAUU replacedSP-UUCACAUCGAUGAAGACAAACUGGAUACAAACUCCGUAUAUGAG withIgKappaCCUUACUACCAUUCAGAUCAUGCGGAGUCUUCAUGGGUAAAUCGGGGAGAGUCUUCGCGAAAAGCGUACGAUCAUAACUCACCUUAUAUAUGGCCACGUAAUGAUUAUGAUGGAUUUUUAGAGAACGCACACGAACACCAUGGGGUGUAUAAUCAGGGCCGUGGUAUCGAUAGCGGGGAACGGUUAAUGCAACCCACACAAAUGUCUGCACAGGAGGAUCUUGGGGACGAUACGGGCAUCCACGUUAUCCCUACGUUAAACGGCGAUGACAGACAUAAAAUUGUAAAUGUGGACCAACGUCAAUACGGUGACGUGUUUAAAGGAGAUCUUAAUCCAAAACCCCAAGGCCAAAGACUCAUUGAGGUGUCAGUGGAAGAAAAUCACCCGUUUACUUUACGCGCACCGAUUCAGCGGAUUUAUGGAGUCCGGUACACCGAGACUUGGAGCUUUUUGCCGUCAUUAACCUGUACGGGAGACGCAGCGCCCGCCAUCCAGCAUAUAUGUUUAAAACAUACAACAUGCUUUCAAGACGUGGUGGUGGAUGUGGAUUGCGCGGAAAAUACUAAAGAGGAUCAGUUGGCCGAAAUCAGUUACCGUUUUCAAGGUAAGAAGGAAGCGGACCAACCGUGGAUUGUUGUAAACACGAGCACACUGUUUGAUGAACUCGAAUUAGACCCCCCCGAGAUUGAACCGGGUGUCUUGAAAGUACUUCGGACAGAAAAACAAUACUUGGGUGUGUACAUUUGGAACAUGCGCGGCUCCGAUGGUACGUCUACCUACGCCACGUUUUUGGUCACCUGGAAAGGGGAUGAAAAAACAAGAAACCCUACGCCCGCAGUAACUCCUCAACCAAGAGGGGCUGAGUUUCAUAUGUGGAAUUACCACUCGCAUGUAUUUUCAGUUGGUGAUACGUUUAGCUUGGCAAUGCAUCUUCAGUAUAAGAUACAUGAAGCGCCAUUUGAUUUGCUGUUAGAGUGGUUGUAUGUCCCCAUCGAUCCUACAUGUCAACCAAUGCGGUUAUAUUCUACGUGUUUGUAUCAUCCCAACGCACCCCAAUGCCUCUCUCAUAUGAAUUCCGGUUGUACAUUUACCUCGCCACAUUUAGCCCAGCGUGUUGCAAGCACAGUGUAUCAAAAUUGUGAACAUGCAGAUAACUACACCGCAUAUUGUCUGGGAAUAUCUCAUAUGGAGCCUAGCUUUGGUCUAAUCUUACACGACGGGGGCACCACGUUAAAGUUUGUAGAUACACCCGAGAGUUUGUCGGGAUUAUACGUUUUUGUGGUGUAUUUUAACGGGCAUGUUGAAGCCGUAGCAUACACUGUUGUAUCCACAGUAGAUCAUUUUGUAAACGCAAUUGAAGAGCGUGGAUUUCCGCCAACGGCCGGUCAGCCACCGGCGACUACUAAACCCAAGGAAAUUACCCCCGUAAACCCCGGAACGUCACCACUUCUACGAUAUGCCGCAUGGACCGGAGGGCUUGCAGCAGUAGUACUUUUAUGUCUCGUAAUAUUUUUAAUCUGUACGGCUUGAUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAUCUAG VZV-GE-G*GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGA 96 full_with_GCCACCAUGGGGACAGUUAAUAAACCUGUGGUGGGGGUAUUGA AEAADAUGGGGUUCGGAAUUAUCACGGGAACGUUGCGUAUAACGAAUCC (SEQ IDGGUCAGAGCAUCCGUCUUGCGAUACGAUGAUUUUCACAUCGAUG NO: 58)AAGACAAACUGGAUACAAACUCCGUAUAUGAGCCUUACUACCAUUCAGAUCAUGCGGAGUCUUCAUGGGUAAAUCGGGGAGAGUCUUCGCGAAAAGCGUACGAUCAUAACUCACCUUAUAUAUGGCCACGUAAUGAUUAUGAUGGAUUUUUAGAGAACGCACACGAACACCAUGGGGUGUAUAAUCAGGGCCGUGGUAUCGAUAGCGGGGAACGGUUAAUGCAACCCACACAAAUGUCUGCACAGGAGGAUCUUGGGGACGAUACGGGCAUCCACGUUAUCCCUACGUUAAACGGCGAUGACAGACAUAAAAUUGUAAAUGUGGACCAACGUCAAUACGGUGACGUGUUUAAAGGAGAUCUUAAUCCAAAACCCCAAGGCCAAAGACUCAUUGAGGUGUCAGUGGAAGAAAAUCACCCGUUUACUUUACGCGCACCGAUUCAGCGGAUUUAUGGAGUCCGGUACACCGAGACUUGGAGCUUUUUGCCGUCAUUAACCUGUACGGGAGACGCAGCGCCCGCCAUCCAGCAUAUAUGUUUAAAACAUACAACAUGCUUUCAAGACGUGGUGGUGGAUGUGGAUUGCGCGGAAAAUACUAAAGAGGAUCAGUUGGCCGAAAUCAGUUACCGUUUUCAAGGUAAGAAGGAAGCGGACCAACCGUGGAUUGUUGUAAACACGAGCACACUGUUUGAUGAACUCGAAUUAGACCCCCCCGAGAUUGAACCGGGUGUCUUGAAAGUACUUCGGACAGAAAAACAAUACUUGGGUGUGUACAUUUGGAACAUGCGCGGCUCCGAUGGUACGUCUACCUACGCCACGUUUUUGGUCACCUGGAAAGGGGAUGAAAAAACAAGAAACCCUACGCCCGCAGUAACUCCUCAACCAAGAGGGGCUGAGUUUCAUAUGUGGAAUUACCACUCGCAUGUAUUUUCAGUUGGUGAUACGUUUAGCUUGGCAAUGCAUCUUCAGUAUAAGAUACAUGAAGCGCCAUUUGAUUUGCUGUUAGAGUGGUUGUAUGUCCCCAUCGAUCCUACAUGUCAACCAAUGCGGUUAUAUUCUACGUGUUUGUAUCAUCCCAACGCACCCCAAUGCCUCUCUCAUAUGAAUUCCGGUUGUACAUUUACCUCGCCACAUUUAGCCCAGCGUGUUGCAAGCACAGUGUAUCAAAAUUGUGAACAUGCAGAUAACUACACCGCAUAUUGUCUGGGAAUAUCUCAUAUGGAGCCUAGCUUUGGUCUAAUCUUACACGACGGGGGCACCACGUUAAAGUUUGUAGAUACACCCGAGAGUUUGUCGGGAUUAUACGUUUUUGUGGUGUAUUUUAACGGGCAUGUUGAAGCCGUAGCAUACACUGUUGUAUCCACAGUAGAUCAUUUUGUAAACGCAAUUGAAGAGCGUGGAUUUCCGCCAACGGCCGGUCAGCCACCGGCGACUACUAAACCCAAGGAAAUUACCCCCGUAAACCCCGGAACGUCACCACUUCUACGAUAUGCCGCAUGGACCGGAGGGCUUGCAGCAGUAGUACUUUUAUGUCUCGUAAUAUUUUUAAUCUGUACGGCUAAACGAAUGAGGGUUAAAGCCUAUAGGGUAGACAAGUCCCCGUAUAACCAAAGCAUGUAUUACGCUGGCCUUCCAGUGGACGAUUUCGAGGACGCCGAAGCCGCCGAUGCCGAAGAAGAGUUUGGUAACGCGAUUGGAGGGAGUCACGGGGGUUCGAGUUACACGGUGUAUAUAGAUAAGACCCGGUGAUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAUCUAG VZV-GE-G*GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGA 97 full_with_GCCACCAUGGGGACAGUUAAUAAACCUGUGGUGGGGGUAUUGA AEAADAUGGGGUUCGGAAUUAUCACGGGAACGUUGCGUAUAACGAAUCC (SEQ IDGGUCAGAGCAUCCGUCUUGCGAUACGAUGAUUUUCACAUCGAUG NO:AAGACAAACUGGAUACAAACUCCGUAUAUGAGCCUUACUACCAU 58)_and_UCAGAUCAUGCGGAGUCUUCAUGGGUAAAUCGGGGAGAGUCUU Y582GCGCGAAAAGCGUACGAUCAUAACUCACCUUAUAUAUGGCCACGUAAUGAUUAUGAUGGAUUUUUAGAGAACGCACACGAACACCAUGGGGUGUAUAAUCAGGGCCGUGGUAUCGAUAGCGGGGAACGGUUAAUGCAACCCACACAAAUGUCUGCACAGGAGGAUCUUGGGGACGAUACGGGCAUCCACGUUAUCCCUACGUUAAACGGCGAUGACAGACAUAAAAUUGUAAAUGUGGACCAACGUCAAUACGGUGACGUGUUUAAAGGAGAUCUUAAUCCAAAACCCCAAGGCCAAAGACUCAUUGAGGUGUCAGUGGAAGAAAAUCACCCGUUUACUUUACGCGCACCGAUUCAGCGGAUUUAUGGAGUCCGGUACACCGAGACUUGGAGCUUUUUGCCGUCAUUAACCUGUACGGGAGACGCAGCGCCCGCCAUCCAGCAUAUAUGUUUAAAACAUACAACAUGCUUUCAAGACGUGGUGGUGGAUGUGGAUUGCGCGGAAAAUACUAAAGAGGAUCAGUUGGCCGAAAUCAGUUACCGUUUUCAAGGUAAGAAGGAAGCGGACCAACCGUGGAUUGUUGUAAACACGAGCACACUGUUUGAUGAACUCGAAUUAGACCCCCCCGAGAUUGAACCGGGUGUCUUGAAAGUACUUCGGACAGAAAAACAAUACUUGGGUGUGUACAUUUGGAACAUGCGCGGCUCCGAUGGUACGUCUACCUACGCCACGUUUUUGGUCACCUGGAAAGGGGAUGAAAAAACAAGAAACCCUACGCCCGCAGUAACUCCUCAACCAAGAGGGGCUGAGUUUCAUAUGUGGAAUUACCACUCGCAUGUAUUUUCAGUUGGUGAUACGUUUAGCUUGGCAAUGCAUCUUCAGUAUAAGAUACAUGAAGCGCCAUUUGAUUUGCUGUUAGAGUGGUUGUAUGUCCCCAUCGAUCCUACAUGUCAACCAAUGCGGUUAUAUUCUACGUGUUUGUAUCAUCCCAACGCACCCCAAUGCCUCUCUCAUAUGAAUUCCGGUUGUACAUUUACCUCGCCACAUUUAGCCCAGCGUGUUGCAAGCACAGUGUAUCAAAAUUGUGAACAUGCAGAUAACUACACCGCAUAUUGUCUGGGAAUAUCUCAUAUGGAGCCUAGCUUUGGUCUAAUCUUACACGACGGGGGCACCACGUUAAAGUUUGUAGAUACACCCGAGAGUUUGUCGGGAUUAUACGUUUUUGUGGUGUAUUUUAACGGGCAUGUUGAAGCCGUAGCAUACACUGUUGUAUCCACAGUAGAUCAUUUUGUAAACGCAAUUGAAGAGCGUGGAUUUCCGCCAACGGCCGGUCAGCCACCGGCGACUACUAAACCCAAGGAAAUUACCCCCGUAAACCCCGGAACGUCACCACUUCUACGAUAUGCCGCAUGGACCGGAGGGCUUGCAGCAGUAGUACUUUUAUGUCUCGUAAUAUUUUUAAUCUGUACGGCUAAACGAAUGAGGGUUAAAGCCUAUAGGGUAGACAAGUCCCCGUAUAACCAAAGCAUGUAUGGCGCUGGCCUUCCAGUGGACGAUUUCGAGGACGCCGAAGCCGCCGAUGCCGAAGAAGAGUUUGGUAACGCGAUUGGAGGGAGUCACGGGGGUUCGAGUUACACGGUGUAUAUAGAUAAGACCCGGUGAUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAUCUAG VZV-GE-G*GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGA 98 Truncated-GCCACCAUGGGGACAGUUAAUAAACCUGUGGUGGGGGUAUUGA delete_from_UGGGGUUCGGAAUUAUCACGGGAACGUUGCGUAUAACGAAUCC 574GGUCAGAGCAUCCGUCUUGCGAUACGAUGAUUUUCACAUCGAUGAAGACAAACUGGAUACAAACUCCGUAUAUGAGCCUUACUACCAUUCAGAUCAUGCGGAGUCUUCAUGGGUAAAUCGGGGAGAGUCUUCGCGAAAAGCGUACGAUCAUAACUCACCUUAUAUAUGGCCACGUAAUGAUUAUGAUGGAUUUUUAGAGAACGCACACGAACACCAUGGGGUGUAUAAUCAGGGCCGUGGUAUCGAUAGCGGGGAACGGUUAAUGCAACCCACACAAAUGUCUGCACAGGAGGAUCUUGGGGACGAUACGGGCAUCCACGUUAUCCCUACGUUAAACGGCGAUGACAGACAUAAAAUUGUAAAUGUGGACCAACGUCAAUACGGUGACGUGUUUAAAGGAGAUCUUAAUCCAAAACCCCAAGGCCAAAGACUCAUUGAGGUGUCAGUGGAAGAAAAUCACCCGUUUACUUUACGCGCACCGAUUCAGCGGAUUUAUGGAGUCCGGUACACCGAGACUUGGAGCUUUUUGCCGUCAUUAACCUGUACGGGAGACGCAGCGCCCGCCAUCCAGCAUAUAUGUUUAAAACAUACAACAUGCUUUCAAGACGUGGUGGUGGAUGUGGAUUGCGCGGAAAAUACUAAAGAGGAUCAGUUGGCCGAAAUCAGUUACCGUUUUCAAGGUAAGAAGGAAGCGGACCAACCGUGGAUUGUUGUAAACACGAGCACACUGUUUGAUGAACUCGAAUUAGACCCCCCCGAGAUUGAACCGGGUGUCUUGAAAGUACUUCGGACAGAAAAACAAUACUUGGGUGUGUACAUUUGGAACAUGCGCGGCUCCGAUGGUACGUCUACCUACGCCACGUUUUUGGUCACCUGGAAAGGGGAUGAAAAAACAAGAAACCCUACGCCCGCAGUAACUCCUCAACCAAGAGGGGCUGAGUUUCAUAUGUGGAAUUACCACUCGCAUGUAUUUUCAGUUGGUGAUACGUUUAGCUUGGCAAUGCAUCUUCAGUAUAAGAUACAUGAAGCGCCAUUUGAUUUGCUGUUAGAGUGGUUGUAUGUCCCCAUCGAUCCUACAUGUCAACCAAUGCGGUUAUAUUCUACGUGUUUGUAUCAUCCCAACGCACCCCAAUGCCUCUCUCAUAUGAAUUCCGGUUGUACAUUUACCUCGCCACAUUUAGCCCAGCGUGUUGCAAGCACAGUGUAUCAAAAUUGUGAACAUGCAGAUAACUACACCGCAUAUUGUCUGGGAAUAUCUCAUAUGGAGCCUAGCUUUGGUCUAAUCUUACACGACGGGGGCACCACGUUAAAGUUUGUAGAUACACCCGAGAGUUUGUCGGGAUUAUACGUUUUUGUGGUGUAUUUUAACGGGCAUGUUGAAGCCGUAGCAUACACUGUUGUAUCCACAGUAGAUCAUUUUGUAAACGCAAUUGAAGAGCGUGGAUUUCCGCCAACGGCCGGUCAGCCACCGGCGACUACUAAACCCAAGGAAAUUACCCCCGUAAACCCCGGAACGUCACCACUUCUACGAUAUGCCGCAUGGACCGGAGGGCUUGCAGCAGUAGUACUUUUAUGUCUCGUAAUAUUUUUAAUCUGUACGGCUAAACGAAUGAGGGUUAAAGCCUAUAGGGUAGACAAGUGAUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAUCUAG VZV-GE-G*GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGA 99 Truncated-GCCACCAUGGGGACAGUUAAUAAACCUGUGGUGGGGGUAUUGA delete_from_UGGGGUUCGGAAUUAUCACGGGAACGUUGCGUAUAACGAAUCC 574_-_GGUCAGAGCAUCCGUCUUGCGAUACGAUGAUUUUCACAUCGAUG Y569AAAGACAAACUGGAUACAAACUCCGUAUAUGAGCCUUACUACCAUUCAGAUCAUGCGGAGUCUUCAUGGGUAAAUCGGGGAGAGUCUUCGCGAAAAGCGUACGAUCAUAACUCACCUUAUAUAUGGCCACGUAAUGAUUAUGAUGGAUUUUUAGAGAACGCACACGAACACCAUGGGGUGUAUAAUCAGGGCCGUGGUAUCGAUAGCGGGGAACGGUUAAUGCAACCCACACAAAUGUCUGCACAGGAGGAUCUUGGGGACGAUACGGGCAUCCACGUUAUCCCUACGUUAAACGGCGAUGACAGACAUAAAAUUGUAAAUGUGGACCAACGUCAAUACGGUGACGUGUUUAAAGGAGAUCUUAAUCCAAAACCCCAAGGCCAAAGACUCAUUGAGGUGUCAGUGGAAGAAAAUCACCCGUUUACUUUACGCGCACCGAUUCAGCGGAUUUAUGGAGUCCGGUACACCGAGACUUGGAGCUUUUUGCCGUCAUUAACCUGUACGGGAGACGCAGCGCCCGCCAUCCAGCAUAUAUGUUUAAAACAUACAACAUGCUUUCAAGACGUGGUGGUGGAUGUGGAUUGCGCGGAAAAUACUAAAGAGGAUCAGUUGGCCGAAAUCAGUUACCGUUUUCAAGGUAAGAAGGAAGCGGACCAACCGUGGAUUGUUGUAAACACGAGCACACUGUUUGAUGAACUCGAAUUAGACCCCCCCGAGAUUGAACCGGGUGUCUUGAAAGUACUUCGGACAGAAAAACAAUACUUGGGUGUGUACAUUUGGAACAUGCGCGGCUCCGAUGGUACGUCUACCUACGCCACGUUUUUGGUCACCUGGAAAGGGGAUGAAAAAACAAGAAACCCUACGCCCGCAGUAACUCCUCAACCAAGAGGGGCUGAGUUUCAUAUGUGGAAUUACCACUCGCAUGUAUUUUCAGUUGGUGAUACGUUUAGCUUGGCAAUGCAUCUUCAGUAUAAGAUACAUGAAGCGCCAUUUGAUUUGCUGUUAGAGUGGUUGUAUGUCCCCAUCGAUCCUACAUGUCAACCAAUGCGGUUAUAUUCUACGUGUUUGUAUCAUCCCAACGCACCCCAAUGCCUCUCUCAUAUGAAUUCCGGUUGUACAUUUACCUCGCCACAUUUAGCCCAGCGUGUUGCAAGCACAGUGUAUCAAAAUUGUGAACAUGCAGAUAACUACACCGCAUAUUGUCUGGGAAUAUCUCAUAUGGAGCCUAGCUUUGGUCUAAUCUUACACGACGGGGGCACCACGUUAAAGUUUGUAGAUACACCCGAGAGUUUGUCGGGAUUAUACGUUUUUGUGGUGUAUUUUAACGGGCAUGUUGAAGCCGUAGCAUACACUGUUGUAUCCACAGUAGAUCAUUUUGUAAACGCAAUUGAAGAGCGUGGAUUUCCGCCAACGGCCGGUCAGCCACCGGCGACUACUAAACCCAAGGAAAUUACCCCCGUAAACCCCGGAACGUCACCACUUCUACGAUAUGCCGCAUGGACCGGAGGGCUUGCAGCAGUAGUACUUUUAUGUCUCGUAAUAUUUUUAAUCUGUACGGCUAAACGAAUGAGGGUUAAAGCCGCCAGGGUAGACAAGUGAUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAUCUAG VZV-GE-AUGGGGACAGUUAAUAAACCUGUGGUGGGGGUAUUGAUGGGGU 133 Truncated-UCGGAAUUAUCACGGGAACGUUGCGUAUAACGAAUCCGGUCAGA delete_from_GCAUCCGUCUUGCGAUACGAUGAUUUUCACAUCGAUGAAGACAA 574_-_ACUGGAUACAAACUCCGUAUAUGAGCCUUACUACCAUUCAGAUC Y569AAUGCGGAGUCUUCAUGGGUAAAUCGGGGAGAGUCUUCGCGAAA (ORF)AGCGUACGAUAAUCAUAACUCACCUUAUAUAUGGCCACGUAAUGAUUAUGAUGGAUUUUUAGAGAACGCACACGAACACCAUGGGGUGUAUAAUCAGGGCCGUGGUAUCGAUAGCGGGGAACGGUUAAUGCAACCCACACAAAUGUCUGCACAGGAGGAUCUUGGGGACGAUACGGGCAUCCACGUUAUCCCUACGUUAAACGGCGAUGACAGACAUAAAAUUGUAAAUGUGGACCAACGUCAAUACGGUGACGUGUUUAAAGGAGAUCUUAAUCCAAAACCCCAAGGCCAAAGACUCAUUGAGGUGUCAGUGGAAGAAAAUCACCCGUUUACUUUACGCGCACCGAUUCAGCGGAUUUAUGGAGUCCGGUACACCGAGACUUGGAGCUUUUUGCCGUCAUUAACCUGUACGGGAGACGCAGCGCCCGCCAUCCAGCAUAUAUGUUUAAAACAUACAACAUGCUUUCAAGACGUGGUGGUGGAUGUGGAUUGCGCGGAAAAUACUAAAGAGGAUCAGUUGGCCGAAAUCAGUUACCGUUUUCAAGGUAAGAAGGAAGCGGACCAACCGUGGAUUGUUGUAAACACGAGCACACUGUUUGAUGAACUCGAAUUAGACCCCCCCGAGAUUGAACCGGGUGUCUUGAAAGUACUUCGGACAGAAAAACAAUACUUGGGUGUGUACAUUUGGAACAUGCGCGGCUCCGAUGGUACGUCUACCUACGCCACGUUUUUGGUCACCUGGAAAGGGGAUGAAAAAACAAGAAACCCUACGCCCGCAGUAACUCCUCAACCAAGAGGGGCUGAGUUUCAUAUGUGGAAUUACCACUCGCAUGUAUUUUCAGUUGGUGAUACGUUUAGCUUGGCAAUGCAUCUUCAGUAUAAGAUACAUGAAGCGCCAUUUGAUUUGCUGUUAGAGUGGUUGUAUGUCCCCAUCGAUCCUACAUGUCAACCAAUGCGGUUAUAUUCUACGUGUUUGUAUCAUCCCAACGCACCCCAAUGCCUCUCUCAUAUGAAUUCCGGUUGUACAUUUACCUCGCCACAUUUAGCCCAGCGUGUUGCAAGCACAGUGUAUCAAAAUUGUGAACAUGCAGAUAACUACACCGCAUAUUGUCUGGGAAUAUCUCAUAUGGAGCCUAGCUUUGGUCUAAUCUUACACGACGGGGGCACCACGUUAAAGUUUGUAGAUACACCCGAGAGUUUGUCGGGAUUAUACGUUUUUGUGGUGUAUUUUAACGGGCAUGUUGAAGCCGUAGCAUACACUGUUGUAUCCACAGUAGAUCAUUUUGUAAACGCAAUUGAAGAGCGUGGAUUUCCGCCAACGGCCGGUCAGCCACCGGCGACUACUAAACCCAAGGAAAUUACCCCCGUAAACCCCGGAACGUCACCACUUCUACGAUAUGCCGCAUGGACCGGAGGGCUUGCAGCAGUAGUACUUUUAUGUCUCGUAAUAUUUUUAAUCUGUACGGCUAAACGAAUGAGGGUUAAAGCCGCCAGGGUAGACAAGUGAUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUG AGUGGGCGGC VZV-GI-G*GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGA 100 fullGCCACCAUGUUUUUAAUCCAAUGUUUGAUAUCGGCCGUUAUAUUUUACAUACAAGUGACCAACGCUUUGAUCUUCAAGGGCGACCACGUGAGCUUGCAAGUUAACAGCAGUCUCACGUCUAUCCUUAUUCCCAUGCAAAAUGAUAAUUAUACAGAGAUAAAAGGACAGCUUGUCUUUAUUGGAGAGCAACUACCUACCGGGACAAACUAUAGCGGAACACUGGAACUGUUAUACGCGGAUACGGUGGCGUUUUGUUUCCGGUCAGUACAAGUAAUAAGAUACGACGGAUGUCCCCGGAUUAGAACGAGCGCUUUUAUUUCGUGUAGGUACAAACAUUCGUGGCAUUAUGGUAACUCAACGGAUCGGAUAUCAACAGAGCCGGAUGCUGGUGUAAUGUUGAAAAUUACCAAACCGGGAAUAAAUGAUGCUGGUGUGUAUGUACUUCUUGUUCGGUUAGACCAUAGCAGAUCCACCGAUGGUUUCAUUCUUGGUGUAAAUGUAUAUACAGCGGGCUCGCAUCACAACAUUCACGGGGUUAUCUACACUUCUCCAUCUCUACAGAAUGGAUAUUCUACAAGAGCCCUUUUUCAACAAGCUCGUUUGUGUGAUUUACCCGCGACACCCAAAGGGUCCGGUACCUCCCUGUUUCAACAUAUGCUUGAUCUUCGUGCCGGUAAAUCGUUAGAGGAUAACCCUUGGUUACAUGAGGACGUUGUUACGACAGAAACUAAGUCCGUUGUUAAGGAGGGGAUAGAAAAUCACGUAUAUCCAACGGAUAUGUCCACGUUACCCGAAAAGUCCCUUAAUGAUCCUCCAGAAAAUCUACUUAUAAUUAUUCCUAUAGUAGCGUCUGUCAUGAUCCUCACCGCCAUGGUUAUUGUUAUUGUAAUAAGCGUUAAGCGACGUAGAAUUAAAAAACAUCCAAUUUAUCGCCCAAAUACAAAAACAAGAAGGGGCAUACAAAAUGCGACACCAGAAUCCGAUGUGAUGUUGGAGGCCGCCAUUGCACAACUAGCAACGAUUCGCGAAGAAUCCCCCCCACAUUCCGUUGUAAACCCGUUUGUUAAAUAGUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAUCUAGVZV-GE- G*GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGA 101 Truncated-GCCACCAUGGGCACCGUGAACAAGCCCGUCGUGGGCGUGCUGAU delete_from_GGGCUUCGGCAUCAUCACCGGCACCCUGCGGAUCACCAAUCCUG 574_-_UGCGGGCCAGCGUGCUGAGAUACGACGACUUCCACAUCGACGAG Y569AGACAAGCUGGACACCAACAGCGUGUACGAGCCCUACUACCACAG Variant 1CGACCACGCCGAGAGCAGCUGGGUCAACAGAGGCGAGUCCAGCCGGAAGGCCUACGACCACAACAGCCCCUACAUCUGGCCCCGGAACGACUACGACGGCUUCCUGGAAAAUGCCCACGAGCACCACGGCGUGUACAACCAGGGCAGAGGCAUCGACAGCGGCGAGAGACUGAUGCAGCCCACCCAGAUGAGCGCCCAGGAAGAUCUGGGCGACGACACCGGCAUCCACGUGAUCCCUACCCUGAACGGCGACGACCGGCACAAGAUCGUGAACGUGGACCAGCGGCAGUACGGCGACGUGUUCAAGGGCGACCUGAACCCCAAGCCCCAGGGACAGCGGCUGAUUGAGGUGUCCGUGGAAGAGAACCACCCCUUCACCCUGAGAGCCCCUAUCCAGCGGAUCUACGGCGUGCGCUAUACCGAGACUUGGAGCUUCCUGCCCAGCCUGACCUGUACUGGCGACGCCGCUCCUGCCAUCCAGCACAUCUGCCUGAAGCACACCACCUGUUUCCAGGACGUGGUGGUGGACGUGGACUGCGCCGAGAACACCAAAGAGGACCAGCUGGCCGAGAUCAGCUACCGGUUCCAGGGCAAGAAAGAGGCCGACCAGCCCUGGAUCGUCGUGAACACCAGCACCCUGUUCGACGAGCUGGAACUGGACCCUCCCGAGAUCGAACCCGGGGUGCUGAAGGUGCUGCGGACCGAGAAGCAGUACCUGGGAGUGUACAUCUGGAACAUGCGGGGCAGCGACGGCACCUCUACCUACGCCACCUUCCUCGUGACCUGGAAGGGCGACGAGAAAACCCGGAACCCUACCCCUGCCGUGACCCCUCAGCCUAGAGGCGCCGAGUUUCACAUGUGGAAUUACCACAGCCACGUGUUCAGCGUGGGCGACACCUUCUCCCUGGCCAUGCAUCUGCAGUACAAGAUCCACGAGGCCCCUUUCGACCUGCUGCUGGAAUGGCUGUACGUGCCCAUCGACCCUACCUGCCAGCCCAUGCGGCUGUACUCCACCUGUCUGUACCACCCCAACGCCCCUCAGUGCCUGAGCCACAUGAAUAGCGGCUGCACCUUCACCAGCCCUCACCUGGCUCAGAGGGUGGCCAGCACCGUGUACCAGAAUUGCGAGCACGCCGACAACUACACCGCCUACUGCCUGGGCAUCAGCCACAUGGAACCCAGCUUCGGCCUGAUCCUGCACGAUGGCGGCACCACCCUGAAGUUCGUGGACACCCCUGAGUCCCUGAGCGGCCUGUACGUGUUCGUGGUGUACUUCAACGGCCACGUGGAAGCCGUGGCCUACACCGUGGUGUCCACCGUGGACCACUUCGUGAACGCCAUCGAGGAACGGGGCUUCCCUCCAACUGCUGGACAGCCUCCUGCCACCACCAAGCCCAAAGAAAUCACCCCUGUGAACCCCGGCACCAGCCCACUGCUGCGCUAUGCUGCUUGGACAGGCGGACUGGCUGCUGUGGUGCUGCUGUGCCUCGUGAUUUUCCUGAUCUGCACCGCCAAGCGGAUGAGAGUGAAGGCCGCCAGAGUGGACAAGUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAUCUAG VZV-GE-G*GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGA 102 Truncated-GCCACCAUGGGGACAGUUAAUAAACCUGUGGUGGGGGUAUUGA delete_from_UGGGGUUCGGAAUUAUCACGGGAACGUUGCGUAUAACGAAUCC 574_-_GGUCAGAGCAUCCGUCUUGCGAUACGAUGAUUUUCACAUCGAUG Y569AAAGACAAACUGGAUACAAACUCCGUAUAUGAGCCUUACUACCAU Variant 2UCAGAUCAUGCGGAGUCUUCAUGGGUAAAUCGGGGAGAGUCUUCGCGAAAAGCGUACGAUCAUAACUCACCUUAUAUAUGGCCACGUAAUGAUUAUGAUGGAUUUUUAGAGAACGCACACGAACACCAUGGGGUGUAUAAUCAGGGCCGUGGUAUCGAUAGCGGGGAACGGUUAAUGCAACCCACACAAAUGUCUGCACAGGAGGAUCUUGGGGACGAUACGGGCAUCCACGUUAUCCCUACGUUAAACGGCGAUGACAGACAUAAAAUUGUAAAUGUGGACCAACGUCAAUACGGUGACGUGUUUAAAGGAGAUCUUAAUCCAAAACCCCAAGGCCAAAGACUCAUUGAGGUGUCAGUGGAAGAAAAUCACCCGUUUACUUUACGCGCACCGAUUCAGCGGAUUUAUGGAGUCCGGUACACCGAGACUUGGAGCUUUUUGCCGUCAUUAACCUGUACGGGAGACGCAGCGCCCGCCAUCCAGCAUAUAUGUUUAAAACAUACAACAUGCUUUCAAGACGUGGUGGUGGAUGUGGAUUGCGCGGAAAAUACUAAAGAGGAUCAGUUGGCCGAAAUCAGUUACCGUUUUCAAGGUAAGAAGGAAGCGGACCAACCGUGGAUUGUUGUAAACACGAGCACACUGUUUGAUGAACUCGAAUUAGACCCCCCCGAGAUUGAACCGGGUGUCUUGAAAGUACUUCGGACAGAGAAACAAUACUUGGGUGUGUACAUUUGGAACAUGCGCGGCUCCGAUGGUACGUCUACCUACGCCACGUUUUUGGUCACCUGGAAAGGGGAUGAGAAGACAAGAAACCCUACGCCCGCAGUAACUCCUCAACCAAGAGGGGCUGAGUUUCAUAUGUGGAAUUACCACUCGCAUGUAUUUUCAGUUGGUGAUACGUUUAGCUUGGCAAUGCAUCUUCAGUAUAAGAUACAUGAAGCGCCAUUUGAUUUGCUGUUAGAGUGGUUGUAUGUCCCCAUCGAUCCUACAUGUCAACCAAUGCGGUUAUAUUCUACGUGUUUGUAUCAUCCCAACGCACCCCAAUGCCUCUCUCAUAUGAAUUCCGGUUGUACAUUUACCUCGCCACAUUUAGCCCAGCGUGUUGCAAGCACAGUGUAUCAAAAUUGUGAACAUGCAGAUAACUACACCGCAUAUUGUCUGGGAAUAUCUCAUAUGGAGCCUAGCUUUGGUCUAAUCUUACACGACGGGGGCACCACGUUAAAGUUUGUAGAUACACCCGAGAGUUUGUCGGGAUUAUACGUUUUUGUGGUGUAUUUUAACGGGCAUGUUGAAGCCGUAGCAUACACUGUUGUAUCCACAGUAGAUCAUUUUGUAAACGCAAUUGAAGAGCGUGGAUUUCCGCCAACGGCCGGUCAGCCACCGGCGACUACUAAACCCAAGGAAAUUACCCCCGUAAACCCCGGAACGUCACCACUUCUACGAUAUGCCGCAUGGACCGGAGGGCUUGCAGCAGUAGUACUUUUAUGUCUCGUAAUAUUUUUAAUCUGUACGGCUAAACGAAUGAGGGUUAAAGCCGCCAGGGUAGACAAGUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAUCUAG VZV-GE-G*GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGA 103 Truncated-GCCACCAUGGGGACAGUUAAUAAACCUGUGGUGGGGGUAUUGA delete_from_UGGGGUUCGGAAUUAUCACGGGAACGUUGCGUAUAACGAAUCC 574_-_GGUCAGAGCAUCCGUCUUGCGAUACGAUGAUUUUCACAUCGAUG Y569AAAGACAAACUGGAUACAAACUCCGUAUAUGAGCCUUACUACCAU Variant 3UCAGAUCAUGCGGAGUCUUCAUGGGUAAAUCGGGGAGAGUCUUCGCGAAAAGCGUACGAUCAUAACUCACCUUAUAUAUGGCCACGUAAUGAUUAUGAUGGAUUUUUAGAGAACGCACACGAACACCAUGGGGUGUAUAAUCAGGGCCGUGGUAUCGAUAGCGGGGAACGGUUAAUGCAACCCACACAAAUGUCUGCACAGGAGGAUCUUGGGGACGAUACGGGCAUCCACGUUAUCCCUACGUUAAACGGCGAUGACAGACAUAAAAUUGUAAAUGUGGACCAACGUCAAUACGGUGACGUGUUUAAAGGAGAUCUUAAUCCAAAACCCCAAGGCCAAAGACUCAUUGAGGUGUCAGUGGAAGAAAAUCACCCGUUUACUUUACGCGCACCGAUUCAGCGGAUUUAUGGAGUCCGGUACACCGAGACUUGGAGCUUUUUGCCGUCAUUAACCUGUACGGGAGACGCAGCGCCCGCCAUCCAGCAUAUAUGUUUAAAACAUACAACAUGCUUUCAAGACGUGGUGGUGGAUGUGGAUUGCGCGGAAAAUACUAAAGAGGAUCAGUUGGCCGAAAUCAGUUACCGUUUUCAAGGUAAGAAGGAAGCGGACCAACCGUGGAUUGUUGUAAACACGAGCACACUGUUUGAUGAACUCGAAUUAGACCCACCCGAGAUUGAACCGGGUGUCUUGAAAGUACUUCGGACAGAGAAACAAUACUUGGGUGUGUACAUUUGGAACAUGCGCGGCUCCGAUGGUACGUCUACCUACGCCACGUUUUUGGUCACCUGGAAAGGGGAUGAGAAGACAAGAAACCCUACGCCCGCAGUAACUCCUCAACCAAGAGGGGCUGAGUUUCAUAUGUGGAAUUACCACUCGCAUGUAUUUUCAGUUGGUGAUACGUUUAGCUUGGCAAUGCAUCUUCAGUAUAAGAUACAUGAAGCGCCAUUUGAUUUGCUGUUAGAGUGGUUGUAUGUCCCCAUCGAUCCUACAUGUCAACCAAUGCGGUUAUAUUCUACGUGUUUGUAUCAUCCCAACGCACCCCAAUGCCUCUCUCAUAUGAAUUCCGGUUGUACAUUUACCUCGCCACAUUUAGCCCAGCGUGUUGCAAGCACAGUGUAUCAAAAUUGUGAACAUGCAGAUAACUACACCGCAUAUUGUCUGGGAAUAUCUCAUAUGGAGCCUAGCUUUGGUCUAAUCUUACACGACGGGGGCACCACGUUAAAGUUUGUAGAUACACCCGAGAGUUUGUCGGGAUUAUACGUUUUUGUGGUGUAUUUUAACGGGCAUGUUGAAGCCGUAGCAUACACUGUUGUAUCCACAGUAGAUCAUUUUGUAAACGCAAUUGAAGAGCGUGGAUUUCCGCCAACGGCCGGUCAGCCACCGGCGACUACUAAACCCAAGGAAAUUACCCCCGUAAACCCCGGAACGUCACCACUUCUACGAUAUGCCGCAUGGACCGGAGGGCUUGCAGCAGUAGUACUUUUAUGUCUCGUAAUAUUUUUAAUCUGUACGGCUAAACGAAUGAGGGUUAAAGCCGCCAGGGUAGACAAGUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAUCUAG VZV-GE-G*GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGA 104 Truncated-GCCACCAUGGGGACAGUUAAUAAACCUGUGGUGGGGGUAUUGA delete_from_UGGGGUUCGGAAUUAUCACGGGAACGUUGCGUAUAACGAAUCC 574_-_GGUCAGAGCAUCCGUCUUGCGAUACGAUGAUUUUCACAUCGAUG Y569AAAGACAAACUGGAUACAAACUCCGUAUAUGAGCCUUACUACCAU Variant 4UCAGAUCAUGCGGAGUCUUCAUGGGUAAAUCGGGGAGAGUCUUCGCGAAAGGCGUACGAUCAUAACUCACCUUAUAUAUGGCCACGUAAUGAUUAUGAUGGAUUUUUAGAGAACGCACACGAACACCAUGGGGUGUAUAAUCAGGGCCGUGGUAUCGAUAGCGGGGAACGGUUAAUGCAACCCACACAAAUGUCUGCACAGGAGGAUCUUGGGGACGAUACGGGCAUCCACGUUAUCCCUACGUUAAACGGCGAUGACAGACAUAAGAUUGUAAAUGUGGACCAACGUCAAUACGGUGACGUGUUUAAAGGAGAUCUUAAUCCAAAGCCCCAAGGCCAAAGACUCAUUGAGGUGUCAGUGGAAGAGAAUCACCCGUUUACUUUACGCGCACCGAUUCAGCGGAUUUAUGGAGUCCGGUACACCGAGACUUGGAGCUUUUUGCCGUCAUUAACCUGUACGGGAGACGCAGCGCCCGCCAUCCAGCAUAUAUGUUUAAAGCAUACAACAUGCUUUCAAGACGUGGUGGUGGAUGUGGAUUGCGCGGAGAAUACUAAAGAGGAUCAGUUGGCCGAAAUCAGUUACCGUUUUCAAGGUAAGAAGGAAGCGGACCAACCGUGGAUUGUUGUAAACACGAGCACACUGUUUGAUGAACUCGAAUUAGACCCCCCCGAGAUUGAACCGGGUGUCUUGAAAGUACUUCGGACAGAGAAACAAUACUUGGGUGUGUACAUUUGGAACAUGCGCGGCUCCGAUGGUACGUCUACCUACGCCACGUUUUUGGUCACCUGGAAAGGGGAUGAGAAGACAAGAAACCCUACGCCCGCAGUAACUCCUCAACCAAGAGGGGCUGAGUUUCAUAUGUGGAAUUACCACUCGCAUGUAUUUUCAGUUGGUGAUACGUUUAGCUUGGCAAUGCAUCUUCAGUAUAAGAUACAUGAAGCGCCAUUUGAUUUGCUGUUAGAGUGGUUGUAUGUCCCCAUCGAUCCUACAUGUCAACCAAUGCGGUUAUAUUCUACGUGUUUGUAUCAUCCCAACGCACCCCAAUGCCUCUCUCAUAUGAAUUCCGGUUGUACAUUUACCUCGCCACAUUUAGCCCAGCGUGUUGCAAGCACAGUGUAUCAGAAUUGUGAACAUGCAGAUAACUACACCGCAUAUUGUCUGGGAAUAUCUCAUAUGGAGCCUAGCUUUGGUCUAAUCUUACACGACGGGGGCACCACGUUAAAGUUUGUAGAUACACCCGAGAGUUUGUCGGGAUUAUACGUUUUUGUGGUGUAUUUUAACGGGCAUGUUGAAGCCGUAGCAUACACUGUUGUAUCCACAGUAGAUCAUUUUGUAAACGCAAUUGAAGAGCGUGGAUUUCCGCCAACGGCCGGUCAGCCACCGGCGACUACUAAACCCAAGGAAAUUACCCCCGUAAACCCCGGAACGUCACCACUUCUACGAUAUGCCGCAUGGACCGGAGGGCUUGCAGCAGUAGUACUUUUAUGUCUCGUAAUAUUUUUAAUCUGUACGGCUAAACGAAUGAGGGUUAAAGCCGCCAGGGUAGACAAGUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAUCUAG VZV-GE-G*GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGA 105 Truncated-GCCACCAUGGGGACAGUUAAUAAACCUGUGGUGGGGGUAUUGA delete_from_UGGGGUUCGGAAUUAUCACGGGAACGUUGCGUAUAACGAAUCC 574_-_GGUCAGAGCAUCCGUCUUGCGAUACGAUGAUUUUCACAUCGAUG Y569AAAGACAAACUGGAUACAAACUCCGUAUAUGAGCCUUACUACCAU Variant 5UCAGAUCAUGCGGAGUCUUCAUGGGUAAAUCGGGGAGAGUCUUCGCGAAAGGCGUACGAUCAUAACUCACCUUAUAUAUGGCCACGUAAUGAUUAUGAUGGAUUUUUAGAGAACGCACACGAACACCAUGGGGUGUAUAAUCAGGGCCGUGGUAUCGAUAGCGGGGAACGGUUAAUGCAACCCACACAAAUGUCUGCACAGGAGGAUCUUGGGGACGAUACGGGCAUCCACGUUAUCCCUACGUUAAACGGCGAUGACAGACAUAAGAUUGUAAAUGUGGACCAACGUCAAUACGGUGACGUGUUUAAAGGAGAUCUUAAUCCAAAGCCCCAAGGCCAAAGACUCAUUGAGGUGUCAGUGGAAGAGAAUCACCCGUUUACUUUACGCGCACCGAUUCAGCGGAUUUAUGGAGUCCGGUACACCGAGACUUGGAGCUUUUUGCCGUCAUUAACCUGUACGGGAGACGCAGCGCCCGCCAUCCAGCAUAUAUGUUUAAAGCAUACAACAUGCUUUCAAGACGUGGUGGUGGAUGUGGAUUGCGCGGAGAAUACUAAAGAGGAUCAGUUGGCCGAAAUCAGUUACCGUUUUCAAGGUAAGAAGGAAGCGGACCAACCGUGGAUUGUUGUAAACACGAGCACACUGUUUGAUGAACUCGAAUUAGACCCACCCGAGAUUGAACCGGGUGUCUUGAAAGUACUUCGGACAGAGAAACAAUACUUGGGUGUGUACAUUUGGAACAUGCGCGGCUCCGAUGGUACGUCUACCUACGCCACGUUUUUGGUCACCUGGAAAGGGGAUGAGAAGACAAGAAACCCUACGCCCGCAGUAACUCCUCAACCAAGAGGGGCUGAGUUUCAUAUGUGGAAUUACCACUCGCAUGUAUUUUCAGUUGGUGAUACGUUUAGCUUGGCAAUGCAUCUUCAGUAUAAGAUACAUGAAGCGCCAUUUGAUUUGCUGUUAGAGUGGUUGUAUGUCCCCAUCGAUCCUACAUGUCAACCAAUGCGGUUAUAUUCUACGUGUUUGUAUCAUCCCAACGCACCCCAAUGCCUCUCUCAUAUGAAUUCCGGUUGUACAUUUACCUCGCCACAUUUAGCCCAGCGUGUUGCAAGCACAGUGUAUCAGAAUUGUGAACAUGCAGAUAACUACACCGCAUAUUGUCUGGGAAUAUCUCAUAUGGAGCCUAGCUUUGGUCUAAUCUUACACGACGGGGGCACCACGUUAAAGUUUGUAGAUACACCCGAGAGUUUGUCGGGAUUAUACGUUUUUGUGGUGUAUUUUAACGGGCAUGUUGAAGCCGUAGCAUACACUGUUGUAUCCACAGUAGAUCAUUUUGUAAACGCAAUUGAAGAGCGUGGAUUUCCGCCAACGGCCGGUCAGCCACCGGCGACUACUAAACCCAAGGAAAUUACCCCCGUAAACCCCGGAACGUCACCACUUCUACGAUAUGCCGCAUGGACCGGAGGGCUUGCAGCAGUAGUACUUUUAUGUCUCGUAAUAUUUUUAAUCUGUACGGCUAAACGAAUGAGGGUUAAAGCCGCCAGGGUAGACAAGUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAUCUAG VZV-GE-G*GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGA 106 Truncated-GCCACCAUGGGGACAGUUAAUAAACCUGUGGUGGGGGUAUUGA delete_from_UGGGGUUCGGAAUUAUCACGGGAACGUUGCGUAUAACGAAUCC 574_-_GGUCAGAGCAUCCGUCUUGCGAUACGAUGAUUUUCACAUCGAUG Y569AAAGACAAACUGGAUACAAACUCCGUAUAUGAGCCUUACUACCAU Variant 6UCAGAUCAUGCGGAGUCUUCAUGGGUAAAUCGGGGAGAGUCUUCGCGAAAAGCGUACGAUCAUAACUCACCUUAUAUAUGGCCACGUAAUGAUUAUGAUGGAUUUUUAGAGAACGCACACGAACACCAUGGGGUGUAUAAUCAGGGCCGUGGUAUCGAUAGCGGGGAACGGUUAAUGCAACCCACACAAAUGUCUGCACAGGAGGAUCUUGGGGACGAUACGGGCAUCCACGUUAUCCCUACGUUAAACGGCGAUGACAGACAUAAAAUUGUAAAUGUGGACCAACGUCAAUACGGUGACGUGUUUAAAGGAGAUCUUAAUCCAAAACCCCAAGGCCAAAGACUCAUUGAGGUGUCAGUGGAAGAAAAUCACCCGUUUACUUUACGCGCACCGAUUCAGCGGAUUUAUGGAGUCCGGUACACCGAGACUUGGAGCUUUUUGCCGUCAUUAACCUGUACGGGAGACGCAGCGCCCGCCAUCCAGCAUAUAUGUUUAAAGCAUACAACAUGCUUUCAAGACGUGGUGGUGGAUGUGGAUUGCGCGGAAAAUACUAAAGAGGAUCAGUUGGCCGAAAUCAGUUACCGUUUUCAAGGUAAGAAGGAAGCGGACCAACCGUGGAUUGUUGUAAACACGAGCACACUGUUUGAUGAACUCGAAUUAGACCCCCCCGAGAUUGAACCGGGUGUCUUGAAAGUACUUCGGACAGAGAAACAAUACUUGGGUGUGUACAUUUGGAACAUGCGCGGCUCCGAUGGUACGUCUACCUACGCCACGUUUUUGGUCACCUGGAAAGGGGAUGAGAAGACAAGAAACCCUACGCCCGCAGUAACUCCUCAACCAAGAGGGGCUGAGUUUCAUAUGUGGAAUUACCACUCGCAUGUAUUUUCAGUUGGUGAUACGUUUAGCUUGGCAAUGCAUCUUCAGUAUAAGAUACAUGAAGCGCCAUUUGAUUUGCUGUUAGAGUGGUUGUAUGUCCCCAUCGAUCCUACAUGUCAACCAAUGCGGUUAUAUUCUACGUGUUUGUAUCAUCCCAACGCACCCCAAUGCCUCUCUCAUAUGAAUUCCGGUUGUACAUUUACCUCGCCACAUUUAGCCCAGCGUGUUGCAAGCACAGUGUAUCAGAAUUGUGAACAUGCAGAUAACUACACCGCAUAUUGUCUGGGAAUAUCUCAUAUGGAGCCUAGCUUUGGUCUAAUCUUACACGACGGGGGCACCACGUUAAAGUUUGUAGAUACACCCGAGAGUUUGUCGGGAUUAUACGUUUUUGUGGUGUAUUUUAACGGGCAUGUUGAAGCCGUAGCAUACACUGUUGUAUCCACAGUAGAUCAUUUUGUAAACGCAAUUGAAGAGCGUGGAUUUCCGCCAACGGCCGGUCAGCCACCGGCGACUACUAAACCCAAGGAAAUUACCCCCGUAAACCCCGGAACGUCACCACUUCUACGAUAUGCCGCAUGGACCGGAGGGCUUGCAGCAGUAGUACUUUUAUGUCUCGUAAUAUUUUUAAUCUGUACGGCUAAACGAAUGAGGGUUAAAGCCGCCAGGGUAGACAAGUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAUCUAG VZV-GE-G*GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGA 136 Truncated-GCCACCAUGGGGACAGUUAAUAAACCUGUGGUGGGGGUAUUGA delete_from_UGGGGUUCGGAAUUAUCACGGGAACGUUGCGUAUAACGAAUCC 574_-_GGUCAGAGCAUCCGUCUUGCGAUACGAUGAUUUUCACAUCGAUG Y569AAAGACAAACUGGAUACAAACUCCGUAUAUGAGCCUUACUACCAU Variant 6UCAGAUCAUGCGGAGUCUUCAUGGGUAAAUCGGGGAGAGUCUU (versionCGCGAAAAGCGUACGAUCAUAACUCACCUUAUAUAUGGCCACGU 2)AAUGAUUAUGAUGGAUUUUUAGAGAACGCACACGAACACCAUGGGGUGUAUAAUCAGGGCCGUGGUAUCGAUAGCGGGGAACGGUUAAUGCAACCCACACAAAUGUCUGCACAGGAGGAUCUUGGGGACGAUACGGGCAUCCACGUUAUCCCUACGUUAAACGGCGAUGACAGACAUAAAAUUGUAAAUGUGGACCAACGUCAAUACGGUGACGUGUUUAAAGGAGAUCUUAAUCCAAAACCCCAAGGCCAAAGACUCAUUGAGGUGUCAGUGGAAGAAAAUCACCCGUUUACUUUACGCGCACCGAUUCAGCGGAUUUAUGGAGUCCGGUACACCGAGACUUGGAGCUUUUUGCCGUCAUUAACCUGUACGGGAGACGCAGCGCCCGCCAUCCAGCAUAUAUGUUUAAAGCAUACAACAUGCUUUCAAGACGUGGUGGUGGAUGUGGAUUGCGCGGAAAAUACUAAAGAGGAUCAGUUGGCCGAAAUCAGUUACCGUUUUCAAGGUAAGAAGGAAGCGGACCAACCGUGGAUUGUUGUAAACACGAGCACACUGUUUGAUGAACUCGAAUUAGACCCCCCCGAGAUUGAACCGGGUGUCUUGAAAGUACUUCGGACAGAGAAACAAUACUUGGGUGUGUACAUUUGGAACAUGCGCGGCUCCGAUGGUACGUCUACCUACGCCACGUUUUUGGUCACCUGGAAAGGGGAUGAGAAGACAAGAAACCCUACGCCCGCAGUAACUCCUCAACCAAGAGGGGCUGAGUUUCAUAUGUGGAAUUACCACUCGCAUGUAUUUUCAGUUGGUGAUACGUUUAGCUUGGCAAUGCAUCUUCAGUAUAAGAUACAUGAAGCGCCAUUUGAUUUGCUGUUAGAGUGGUUGUAUGUCCCCAUCGAUCCUACAUGUCAACCAAUGCGGUUAUAUUCUACGUGUUUGUAUCAUCCCAACGCACCCCAAUGCCUCUCUCAUAUGAAUUCCGGUUGUACAUUUACCUCGCCACAUUUAGCCCAGCGUGUUGCAAGCACAGUGUAUCAGAAUUGUGAACAUGCAGAUAACUACACCGCAUAUUGUCUGGGAAUAUCUCAUAUGGAGCCUAGCUUUGGUCUAAUCUUACACGACGGGGGCACCACGUUAAAGUUUGUAGAUACACCCGAGAGUUUGUCGGGAUUAUACGUUUUUGUGGUGUAUUUUAACGGGCAUGUUGAAGCCGUAGCAUACACUGUUGUAUCCACAGUAGAUCAUUUUGUAAACGCAAUUGAAGAGCGUGGAUUUCCGCCAACGGCCGGUCAGCCACCGGCGACUACUAAACCCAAGGAAAUUACCCCCGUAAACCCCGGAACGUCACCACUUCUACGAUAUGCCGCAUGGACCGGAGGGCUUGCAGCAGUAGUACUUUUAUGUCUCGUAAUAUUUUUAAUCUGUACGGCUAAACGAAUGAGGGUUAAAGCCGCCAGGGUAGACAAGUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAUCUAG VZV-GE-G*GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGA 107 Truncated-GCCACCAUGGGGACAGUUAAUAAACCUGUGGUGGGGGUAUUGA delete_from_UGGGGUUCGGAAUUAUCACGGGAACGUUGCGUAUAACGAAUCC 574_-_GGUCAGAGCAUCCGUCUUGCGAUACGAUGAUUUUCACAUCGAUG Y569AAAGACAAACUGGAUACAAACUCCGUAUAUGAGCCUUACUACCAU Variant 7UCAGAUCAUGCGGAGUCUUCAUGGGUAAAUCGGGGAGAGUCUUCGCGAAAAGCGUACGAUCAUAACUCACCUUAUAUAUGGCCACGUAAUGAUUAUGAUGGAUUUUUAGAGAACGCACACGAACACCAUGGGGUGUAUAAUCAGGGCCGUGGUAUCGAUAGCGGGGAACGGUUAAUGCAACCCACACAAAUGUCUGCACAGGAGGAUCUUGGGGACGAUACGGGCAUCCACGUUAUCCCUACGUUAAACGGCGAUGACAGACAUAAAAUUGUAAAUGUGGACCAACGUCAAUACGGUGACGUGUUUAAAGGAGAUCUUAAUCCAAAACCCCAAGGCCAAAGACUCAUUGAGGUGUCAGUGGAAGAAAAUCACCCGUUUACUUUACGCGCACCGAUUCAGCGGAUUUAUGGAGUCCGGUACACCGAGACUUGGAGCUUUUUGCCGUCAUUAACCUGUACGGGAGACGCAGCGCCCGCCAUCCAGCAUAUAUGUUUAAAGCAUACAACAUGCUUUCAAGACGUGGUGGUGGAUGUGGAUUGCGCGGAAAAUACUAAAGAGGAUCAGUUGGCCGAAAUCAGUUACCGUUUUCAAGGUAAGAAGGAAGCGGACCAACCGUGGAUUGUUGUAAACACGAGCACACUGUUUGAUGAACUCGAAUUAGACCCACCCGAGAUUGAACCGGGUGUCUUGAAAGUACUUCGGACAGAGAAACAAUACUUGGGUGUGUACAUUUGGAACAUGCGCGGCUCCGAUGGUACGUCUACCUACGCCACGUUUUUGGUCACCUGGAAAGGGGAUGAGAAGACAAGAAACCCUACGCCCGCAGUAACUCCUCAACCAAGAGGGGCUGAGUUUCAUAUGUGGAAUUACCACUCGCAUGUAUUUUCAGUUGGUGAUACGUUUAGCUUGGCAAUGCAUCUUCAGUAUAAGAUACAUGAAGCGCCAUUUGAUUUGCUGUUAGAGUGGUUGUAUGUCCCCAUCGAUCCUACAUGUCAACCAAUGCGGUUAUAUUCUACGUGUUUGUAUCAUCCCAACGCACCCCAAUGCCUCUCUCAUAUGAAUUCCGGUUGUACAUUUACCUCGCCACAUUUAGCCCAGCGUGUUGCAAGCACAGUGUAUCAGAAUUGUGAACAUGCAGAUAACUACACCGCAUAUUGUCUGGGAAUAUCUCAUAUGGAGCCUAGCUUUGGUCUAAUCUUACACGACGGGGGCACCACGUUAAAGUUUGUAGAUACACCCGAGAGUUUGUCGGGAUUAUACGUUUUUGUGGUGUAUUUUAACGGGCAUGUUGAAGCCGUAGCAUACACUGUUGUAUCCACAGUAGAUCAUUUUGUAAACGCAAUUGAAGAGCGUGGAUUUCCGCCAACGGCCGGUCAGCCACCGGCGACUACUAAACCCAAGGAAAUUACCCCCGUAAACCCCGGAACGUCACCACUUCUACGAUAUGCCGCAUGGACCGGAGGGCUUGCAGCAGUAGUACUUUUAUGUCUCGUAAUAUUUUUAAUCUGUACGGCUAAACGAAUGAGGGUUAAAGCCGCCAGGGUAGACAAGUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAUCUAG VZV-GE-AUGGGGACAGUUAAUAAACCUGUGGUGGGGGUAUUGAUGGGGU 134 Truncated-UCGGAAUUAUCACGGGAACGUUGCGUAUAACGAAUCCGGUCAGA delete_from_GCAUCCGUCUUGCGAUACGAUGAUUUUCACAUCGAUGAAGACAA 574_-_ACUGGAUACAAACUCCGUAUAUGAGCCUUACUACCAUUCAGAUC Y569AAUGCGGAGUCUUCAUGGGUAAAUCGGGGAGAGUCUUCGCGAAA Variant 7AGCGUACGAUCAUAACUCACCUUAUAUAUGGCCACGUAAUGAUUAUGAUGGAUUUUUAGAGAACGCACACGAACACCAUGGGGUGUAUAAUCAGGGCCGUGGUAUCGAUAGCGGGGAACGGUUAAUGCAACCCACACAAAUGUCUGCACAGGAGGAUCUUGGGGACGAUACGGGCAUCCACGUUAUCCCUACGUUAAACGGCGAUGACAGACAUAAAAUUGUAAAUGUGGACCAACGUCAAUACGGUGACGUGUUUAAAGGAGAUCUUAAUCCAAAACCCCAAGGCCAAAGACUCAUUGAGGUGUCAGUGGAAGAAAAUCACCCGUUUACUUUACGCGCACCGAUUCAGCGGAUUUAUGGAGUCCGGUACACCGAGACUUGGAGCUUUUUGCCGUCAUUAACCUGUACGGGAGACGCAGCGCCCGCCAUCCAGCAUAUAUGUUUAAAGCAUACAACAUGCUUUCAAGACGUGGUGGUGGAUGUGGAUUGCGCGGAAAAUACUAAAGAGGAUCAGUUGGCCGAAAUCAGUUACCGUUUUCAAGGUAAGAAGGAAGCGGACCAACCGUGGAUUGUUGUAAACACGAGCACACUGUUUGAUGAACUCGAAUUAGACCCACCCGAGAUUGAACCGGGUGUCUUGAAAGUACUUCGGACAGAGAAACAAUACUUGGGUGUGUACAUUUGGAACAUGCGCGGCUCCGAUGGUACGUCUACCUACGCCACGUUUUUGGUCACCUGGAAAGGGGAUGAGAAGACAAGAAACCCUACGCCCGCAGUAACUCCUCAACCAAGAGGGGCUGAGUUUCAUAUGUGGAAUUACCACUCGCAUGUAUUUUCAGUUGGUGAUACGUUUAGCUUGGCAAUGCAUCUUCAGUAUAAGAUACAUGAAGCGCCAUUUGAUUUGCUGUUAGAGUGGUUGUAUGUCCCCAUCGAUCCUACAUGUCAACCAAUGCGGUUAUAUUCUACGUGUUUGUAUCAUCCCAACGCACCCCAAUGCCUCUCUCAUAUGAAUUCCGGUUGUACAUUUACCUCGCCACAUUUAGCCCAGCGUGUUGCAAGCACAGUGUAUCAGAAUUGUGAACAUGCAGAUAACUACACCGCAUAUUGUCUGGGAAUAUCUCAUAUGGAGCCUAGCUUUGGUCUAAUCUUACACGACGGGGGCACCACGUUAAAGUUUGUAGAUACACCCGAGAGUUUGUCGGGAUUAUACGUUUUUGUGGUGUAUUUUAACGGGCAUGUUGAAGCCGUAGCAUACACUGUUGUAUCCACAGUAGAUCAUUUUGUAAACGCAAUUGAAGAGCGUGGAUUUCCGCCAACGGCCGGUCAGCCACCGGCGACUACUAAACCCAAGGAAAUUACCCCCGUAAACCCCGGAACGUCACCACUUCUACGAUAUGCCGCAUGGACCGGAGGGCUUGCAGCAGUAGUACUUUUAUGUCUCGUAAUAUUUUUAAUCUGUACGGCUAAACGAAUGAGGGUUAAAGCCGCCAGGGUAGACAAGUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGG GCGGC VZV-GE-G*AGAAGAAAUAUAAGAGCCACCAUGGGGACAGUUAAUAAACCU 108 Truncated-GUGGUGGGCGUAUUGAUGGGGUUCGGAAUUAUCACGGGAACGU delete_from_UGCGUAUAACGAAUCCGGUCAGAGCAUCCGUCUUGCGAUACGAU 574_-_GAUUUUCACAUCGAUGAAGACAAACUGGAUACAAACUCCGUAUA Y569AUGAGCCUUACUACCAUUCAGAUCAUGCGGAGUCUUCAUGGGUAA Variant 8AUCGGGGAGAGUCUUCGCGAAAGGCGUACGAUCAUAACUCACCUUAUAUAUGGCCACGUAAUGAUUAUGAUGGAUUCUUAGAGAACGCACACGAACACCAUGGGGUGUAUAAUCAGGGCCGUGGUAUCGAUAGCGGGGAACGGUUAAUGCAACCCACACAAAUGUCUGCACAGGAGGAUCUUGGGGACGAUACGGGCAUCCACGUUAUCCCUACGUUAAACGGCGAUGACAGACAUAAGAUUGUAAAUGUGGACCAACGUCAAUACGGUGACGUGUUUAAAGGAGAUCUUAAUCCAAAGCCCCAAGGCCAAAGACUCAUUGAGGUGUCAGUGGAAGAGAAUCACCCGUUUACUUUACGCGCACCGAUUCAGCGGAUUUAUGGAGUCCGGUACACCGAGACUUGGAGCUUCUUGCCGUCAUUAACCUGUACGGGAGACGCAGCGCCCGCCAUCCAGCAUAUAUGUUUAAAGCAUACAACAUGCUUUCAAGACGUGGUGGUGGAUGUGGAUUGCGCGGAGAAUACUAAAGAGGAUCAGUUGGCCGAAAUCAGUUACCGUUUUCAAGGUAAGAAGGAAGCGGACCAACCGUGGAUUGUUGUAAACACGAGCACACUGUUUGAUGAACUCGAAUUAGACCCACCCGAGAUUGAACCGGGUGUCUUGAAAGUACUUCGGACAGAGAAACAAUACUUGGGUGUGUACAUUUGGAACAUGCGCGGCUCCGAUGGUACGUCUACCUACGCCACGUUCUUGGUCACCUGGAAAGGGGAUGAGAAGACAAGAAACCCUACGCCCGCAGUAACUCCUCAACCAAGAGGGGCUGAGUUUCAUAUGUGGAAUUACCACUCGCAUGUAUUUUCAGUUGGUGAUACGUUUAGCUUGGCAAUGCAUCUUCAGUAUAAGAUACAUGAAGCGCCAUUUGAUUUGCUGUUAGAGUGGUUGUAUGUCCCCAUCGAUCCUACAUGUCAACCAAUGCGGUUAUAUUCUACGUGUUUGUAUCAUCCCAACGCACCCCAAUGCCUCUCUCAUAUGAAUUCCGGUUGUACAUUUACCUCGCCACAUUUAGCCCAGCGUGUUGCAAGCACAGUGUAUCAGAAUUGUGAACAUGCAGAUAACUACACCGCAUAUUGUCUGGGAAUAUCUCAUAUGGAGCCUAGCUUUGGUCUAAUCUUACACGACGGAGGCACCACGUUAAAGUUUGUAGAUACACCCGAGAGUUUGUCGGGAUUAUACGUCUUUGUGGUGUAUUUUAACGGGCAUGUUGAAGCCGUAGCAUACACUGUUGUAUCCACAGUAGAUCAUUUUGUAAACGCAAUUGAAGAGCGUGGAUUUCCGCCAACGGCCGGUCAGCCACCGGCGACUACUAAACCCAAGGAAAUUACGCCCGUAAACCCCGGAACGUCACCACUUCUACGAUAUGCCGCAUGGACCGGAGGGCUUGCAGCAGUAGUACUUUUAUGUCUCGUAAUAUUCUUAAUCUGUACGGCUAAACGAAUGAGGGUUAAAGCCGCCAGGGUAGACAAGUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAUCUAG VZV-GE-G*GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGA 141 Truncated-GCCACCAUGGGGACAGUUAAUAAACCUGUGGUGGGCGUAUUGA delete_from_UGGGGUUCGGAAUUAUCACGGGAACGUUGCGUAUAACGAAUCC 574_-_GGUCAGAGCAUCCGUCUUGCGAUACGAUGAUUUUCACAUCGAUG Y569AAAGACAAACUGGAUACAAACUCCGUAUAUGAGCCUUACUACCAU Variant 8UCAGAUCAUGCGGAGUCUUCAUGGGUAAAUCGGGGAGAGUCUU (5′UTRCGCGAAAGGCGUACGAUCAUAACUCACCUUAUAUAUGGCCACGU includesAAUGAUUAUGAUGGAUUCUUAGAGAACGCACACGAACACCAUG promoterGGGUGUAUAAUCAGGGCCGUGGUAUCGAUAGCGGGGAACGGUU region)AAUGCAACCCACACAAAUGUCUGCACAGGAGGAUCUUGGGGACGAUACGGGCAUCCACGUUAUCCCUACGUUAAACGGCGAUGACAGACAUAAGAUUGUAAAUGUGGACCAACGUCAAUACGGUGACGUGUUUAAAGGAGAUCUUAAUCCAAAGCCCCAAGGCCAAAGACUCAUUGAGGUGUCAGUGGAAGAGAAUCACCCGUUUACUUUACGCGCACCGAUUCAGCGGAUUUAUGGAGUCCGGUACACCGAGACUUGGAGCUUCUUGCCGUCAUUAACCUGUACGGGAGACGCAGCGCCCGCCAUCCAGCAUAUAUGUUUAAAGCAUACAACAUGCUUUCAAGACGUGGUGGUGGAUGUGGAUUGCGCGGAGAAUACUAAAGAGGAUCAGUUGGCCGAAAUCAGUUACCGUUUUCAAGGUAAGAAGGAAGCGGACCAACCGUGGAUUGUUGUAAACACGAGCACACUGUUUGAUGAACUCGAAUUAGACCCACCCGAGAUUGAACCGGGUGUCUUGAAAGUACUUCGGACAGAGAAACAAUACUUGGGUGUGUACAUUUGGAACAUGCGCGGCUCCGAUGGUACGUCUACCUACGCCACGUUCUUGGUCACCUGGAAAGGGGAUGAGAAGACAAGAAACCCUACGCCCGCAGUAACUCCUCAACCAAGAGGGGCUGAGUUUCAUAUGUGGAAUUACCACUCGCAUGUAUUUUCAGUUGGUGAUACGUUUAGCUUGGCAAUGCAUCUUCAGUAUAAGAUACAUGAAGCGCCAUUUGAUUUGCUGUUAGAGUGGUUGUAUGUCCCCAUCGAUCCUACAUGUCAACCAAUGCGGUUAUAUUCUACGUGUUUGUAUCAUCCCAACGCACCCCAAUGCCUCUCUCAUAUGAAUUCCGGUUGUACAUUUACCUCGCCACAUUUAGCCCAGCGUGUUGCAAGCACAGUGUAUCAGAAUUGUGAACAUGCAGAUAACUACACCGCAUAUUGUCUGGGAAUAUCUCAUAUGGAGCCUAGCUUUGGUCUAAUCUUACACGACGGAGGCACCACGUUAAAGUUUGUAGAUACACCCGAGAGUUUGUCGGGAUUAUACGUCUUUGUGGUGUAUUUUAACGGGCAUGUUGAAGCCGUAGCAUACACUGUUGUAUCCACAGUAGAUCAUUUUGUAAACGCAAUUGAAGAGCGUGGAUUUCCGCCAACGGCCGGUCAGCCACCGGCGACUACUAAACCCAAGGAAAUUACGCCCGUAAACCCCGGAACGUCACCACUUCUACGAUAUGCCGCAUGGACCGGAGGGCUUGCAGCAGUAGUACUUUUAUGUCUCGUAAUAUUCUUAAUCUGUACGGCUAAACGAAUGAGGGUUAAAGCCGCCAGGGUAGACAAGUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAUCUAG VZV-GE-G*GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGA 137 Truncated-GCCACCAUGGGCACCGUGAACAAGCCUGUUGUGGGCGUGCUGAU delete_from_GGGCUUCGGCAUCAUCACAGGCACCCUGCGGAUCACCAAUCCUG 574_-_UGCGGGCUAGCGUGCUGAGAUACGACGACUUCCACAUCGACGAG Y569AGACAAGCUGGACACCAACAGCGUGUACGAGCCCUACUACCACAG Variant 9CGAUCACGCCGAGUCUAGCUGGGUCAACAGAGGCGAGAGCAGCAGAAAGGCCUACGACCACAACAGCCCUUACAUCUGGCCCAGAAACGACUACGACGGCUUCCUCGAGAAUGCCCACGAACACCACGGCGUGUACAAUCAAGGCAGAGGCAUCGACAGCGGCGAGAGACUGAUGCAGCCUACACAGAUGAGCGCCCAAGAGGACCUGGGAGAUGAUACCGGCAUCCACGUGAUCCCUACACUGAACGGCGACGACCGGCACAAGAUCGUGAACGUGGACCAGAGACAGUACGGCGACGUGUUCAAGGGCGACCUGAAUCCUAAGCCUCAGGGCCAGCGCCUGAUCGAGGUUUCCGUGGAAGAGAAUCACCCUUUCACACUGCGGGCUCCCAUCCAGAGAAUCUACGGCGUGCGCUAUACCGAGACAUGGUCCUUUCUGCCCAGCCUGACAUGUACCGGCGACGCCGCUCCUGCCAUCCAGCACAUUUGUCUGAAGCACACCACCUGUUUCCAGGACGUGGUGGUGGAUGUGGACUGCGCCGAGAACACCAAAGAGGAUCAGCUGGCCGAGAUCAGCUACCGGUUCCAGGGAAAGAAAGAGGCCGACCAGCCUUGGAUCGUGGUCAACACCAGCACACUGUUCGACGAGCUGGAACUGGACCCUCCUGAGAUUGAACCCGGCGUCCUGAAGGUGCUGAGAACCGAGAAGCAGUACCUGGGAGUGUACAUCUGGAACAUGAGAGGCAGCGACGGCACCUCUACCUACGCCACCUUUCUGGUCACAUGGAAGGGCGACGAGAAGACCAGAAAUCCCACACCAGCCGUGACACCUCAGCCUAGAGGCGCCGAAUUUCACAUGUGGAACUACCACUCUCACGUGUUCAGCGUGGGCGAUACCUUCAGCCUGGCCAUGCAUCUGCAGUACAAGAUCCACGAGGCUCCCUUCGACCUGCUGCUGGAAUGGCUGUACGUGCCCAUCGAUCCUACCUGCCAGCCUAUGCGGCUGUACUCCACCUGUCUGUAUCACCCUAACGCUCCUCAGUGCCUGAGCCACAUGAAUAGCGGCUGCACCUUCACAAGCCCUCACCUGGCUCAGCGAGUGGCCAGCACAGUGUACCAGAAUUGCGAGCACGCCGACAAUUACACCGCCUACUGUCUGGGCAUCAGCCACAUGGAACCUAGCUUCGGCCUGAUCCUGCACGAUGGCGGCACCACACUGAAGUUCGUGGACACACCUGAGAGCCUGAGCGGCCUGUAUGUGUUUGUGGUGUACUUCAACGGCCACGUGGAAGCCGUGGCCUACACCGUGGUGUCUACCGUGGACCACUUCGUGAACGCCAUCGAGGAAAGAGGCUUCCCUCCAACUGCUGGACAGCCUCCUGCCACCACCAAGCCUAAAGAAAUCACACCCGUGAAUCCCGGCACUAGCCCUCUGCUUAGAUACGCCGCUUGGACAGGCGGACUGGCUGCUGUUGUUCUGCUGUGCCUGGUCAUCUUCCUGAUCUGCACCGCCAAGCGGAUGAGAGUGAAAGCCGCCAGAGUGGACAAGUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAUCUAG G*represents a 5′ terminal cap, e.g., 7mG(5′)ppp(5′)NlmpNp All mRNAscontains a 5′-UTR, a 3′UTR, and a polyA tail. 5′-UTR:GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCCACC (SEQ ID NO: 138) 3′-UTR:UGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC(SEQ ID NO: 139) polyA tail:AAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAUCUAG(SEQ ID NO: 140)

It should also be understood that the 5′ and/or 3′ UTR for eachconstruct may be omitted, modified or substituted for a different UTRsequences in any one of the vaccines as provided herein.

Example 14: Variant gE Antigen Distribution in Vero and Mewo Cells

The expression and trafficking of VZV gE antigens having different Cterminal variants was investigated in Vero cells and Mewo cells.

Vero cells are lineages of cells used in cell cultures. The ‘Vero’lineage was isolated from kidney epithelial cells extracted from anAfrican green monkey. MeWo cells are human malignant melanoma cells thatare susceptible to VZV infection. Vero cells or Mewo cells weretransfected with the constructs indicated below in Table 3. Thetransfected cells were stained with antibodies for gE and for golgimarkers GM 130 and golgin. Confocal microscopy was used to visualize thestained cells. The results for the constructs are described in Table 3(“Cellular localization” column). FIG. 9 provides an exemplaryexperiment, which shows the results of the following transfectedconstructs: (1) VZV gE mRNA encoding a VZV gE polypeptide having a 62amino acid deletion at the C-terminus (encoded by SEQ ID NO: 3); (2)full-length VZV gE mRNA encoding a VZV gE polypeptide having the AEAADAsequence (SEQ ID NO: 58) (encoded by SEQ ID NO: 7); or (3) PBS (asnegative control). Using an anti-gE antibody, FIG. 9 shows that thetruncated VZV gE polypeptide (having the 62 amino acid C-terminaldeletion) localizes to a perinuclear location and organelles. Thefull-length VZV gE polypeptide having AEAADA sequence (SEQ ID NO: 58)was localized to the golgi and a perinuclear location. Importantly,several of the constructs, e.g., gE-truncated-delete_from_574_Y569A, gEfull length with AEAADA (SEQ ID NO: 58), gE full length with AEAADA (SEQID NO: 58) and Y582C mutation, gE-truncated-delete_from_574, andgE-truncated-delete_from_574 with Y569A mutation each encodedpolypeptides that localized to the cell membrane, indicating that thesepolypeptides may have enhanced antigenicity.

TABLE 3 Summary of Results for Cellular Trafficking of Variant VZV gEPolypeptides Experimental Cellular Construct conditions Expressionlocalization Full length gE Vero cells- 500 ng/well, + shows Golgitransfected 24 h localization transfection (C8) Construct = Full lengthGE-full with GE Vero cells- 500 ng/well, shows Golgi AEAADA transfected24 h +++ localization (SEQ ID transfection (C1)- and diffuse NO: 58)Construct = VZV-GE- perinuclear full with AEAADA (SEQ ID NO: 58) GE-fullwith Vero cells- 500 ng/well, low shows AEAADA transfected organelles(SEQ ID 24 h transfection and NO: 58) (C6) Construct = VZV-GEcytoplasmic and Y582C full_with_AEAADA localization (SEQ ID NO:58)_and_Y582G GE-delete-562 Vero cells- 500 ng/well, + shows transfectedperinuclear 24 h transfection and (C2)-Construct = C2 VZV- organellesGE-delete-562 GE-delete-562- Vero cells- 500 ng/well, +++ shows replacedSP- transfected golgi with 24 h transfection localization IgKappa (C5)VZV-GE-delete- and 562-replacedSignal cytoplasmic Peptide-with IgKappaGE-truncated- Vero cells- 500 ng/well, ++ shows delete_from_574transfected 24 h Golgi transfection (C4) - and Construct = VZV-GE-cytoplasmic truncated-delete_from_574 localization GE-truncated- Verocells- 500 ng/well, +++ shows delete_from_ transfected Golgi 574_Y569A24 h transfection (C3)- and cell Construct = VZV-GE- membranetruncated-delete_ localization from_574_Y569A Full length gE MeWo cells-500 ng/well, +++ shows transfected Golgi 24 h transfection (C8)localization Construct = Full length GE GE-full with MeWo cells- 500ng/well, +++ shows AEAADA transfected 24 h Golgi (SEQ transfection (C1)-and ID NO: 58) Construct = VZV-GE- Membrane full with AEAADAlocalization (SEQ ID NO: 58) GE-full with MeWo cells- 500 ng/well, ++shows AEAADA transfected 24 h golgi (SEQ transfection (C6) and cell IDNO: 58) Construct = SE-VZV- membrane and Y582C GE full_with_AEAADAlocalization (SEQ ID NO: 58)_and_Y582G GE-delete-562 MeWo cells- 500ng/well, +++ shows transfected 24 h perinuclear transfection and (C2)-Construct = C2 cytoplasmic VZV-GE-delete-562 localization GE-delete-562-MeWo cells- 500 ng/well, +++ shows replaced SP- transfected golgi with24 h transfection localization Ig Kappa (C5) VZV-GE-delete-562- andreplacedSignal cytoplasmic Peptide with IgKappa GE-truncated- MeWocells- 500 ng/well, +++ shows delete_from_574 transfected 24 h Golgitransfection (C4) - and cell Construct = -VZV-GE- membranetruncated-delete_from_574 localization GE-truncated- MeWo cells- 500ng/well, +++ shows delete_ transfected Golgi from_574_ 24 h transfectionand cell Y569A (C3)- Construct = membrane VZV-GE-truncated- localizationdelete_from_574_Y569A

Example 15: Immunization of BALB/C Mice with MC3 Formulated mRNA EncodedVZV gE Antigens

An immunization study was conducted as an initial evaluation of theeffect of MC3-formulated mRNAs encoding VZV antigens as vaccinecandidates to achieve immunization in BALB/C mice post intramuscular orintradermal administration.

The candidate vaccines were as follows:

(1) MC3 formulated VZVgE-hIg kappa mRNA having 5′ cap:m7G(5′)ppp(5′)G-2′-O-methyl, N1-methylpseudouridine chemicalmodification, and the additional hIg Kappa sequence.

(2) MC3 formulated VZV gE mRNA having 5′ cap:m7G(5′)ppp(5′)G-2′-O-methyl and N1-methylpseudouridine chemicalmodification.

(3) MC3 formulated VZVgE mRNA having 5′ cap: m7G(5′)ppp(5′)G-2′-O-methyland no chemical modification.

All of the VZV gE mRNAs were strain Oka.

BALB/C mice were given a single 10 μg dose or two 10 μg doses (at day28) of MC3 formulated VZV gE mRNA (either vaccine (1), (2), or (3)described above) either intramuscularly or intradermally. G5 refers mRNAhaving N1-methylpseudouridine chemical modification. GO refers tounmodified mRNA. Cap 1 refers to 5′ cap: m7G(5′)ppp(5′)G-2′-O-methyl.Each treatment group contained eight mice. The positive control wasVARIVAX® vaccine and the negative control was PBS.

Blood samples were taken to determine the presence/level of serumprotein and antibodies. Western blots were performed to detect VZV-gEprotein expression at six hours and ELISAs were performed to detectmouse IgGs. Schematics of the constructs encoding VZV gE (strain Oka)are shown in FIG. 2. Schematics of the study's design and schedule ofinjection are shown in FIG. 3 and Table 3. Table 4 shows the varioustime points for collection of different samples. Blood was collected forserum protein and antibody determination, while VZV protein expressionwas surveyed 6 hours post-dosing on day 0 for groups 1-4, 13, and 14,and 6 hours post-dosing on day 28 for groups 2, 4, and 14. Antibodydetection assays were performed on day −3, day 14, day 27, day 42, andday 56.

TABLE 4 Injection Schedule Dose mRNA Dose Vol 1^(st) 2^(nd) Conc.Volume + G# Antigen Route N = (μg) (μl) dose dose LNP (mg/ml) Overage 1VZV-gE-oka- IM 8 10 50 Day 0 MC3 0.2 1 × 600 μl hIgkappa (G5; cap 1)(SEQ ID NO: 93) 2 VZV-gE-oka- IM 8 10 50 Day 0 Day MC3 0.2 2 × 600 μlhIgkappa (G5; 28 cap 1) 3 VZV-gE-oka- ID 8 10 50 Day 0 MC3 0.2 1 × 600μl hIgkappa (G5; cap 1) 4 VZV-gE-oka- ID 8 10 50 Day 0 Day MC3 0.2 2 ×600 μl hIgkappa (G5; 28 cap 1) 5 VZV-gE-oka IM 8 10 50 Day 0 MC3 0.2 1 ×600 μl (G0; cap 1) (SEQ ID NO: 92) 6 VZV-gE-oka IM 8 10 50 Day 0 Day MC30.2 2 × 600 μl (G0; cap1) 28 7 VZV-gE-oka ID 8 10 50 Day 0 MC3 0.2 1 ×600 μl (G0; cap1) 8 VZV-gE-oka ID 8 10 50 Day 0 Day MC3 0.2 2 × 600 μl(G0; cap1) 28 9 VZV-gE-oka IM 8 10 50 Day 0 MC3 0.2 1 × 600 μl (G5;cap1) 10 VZV-gE-oka IM 8 10 50 Day 0 Day MC3 0.2 2 × 600 μl (G5; cap 1)28 11 VZV-gE-oka ID 8 10 50 Day 0 MC3 0.2 1 × 600 μl (G5; cap1) 12VZV-gE-oka ID 8 10 50 Day 0 Day MC3 0.2 2 × 600 μl (G5; cap1) 28 13Negative IM 6 / 50 Day 0 PBS / 1 × 600 μl control (PBS) 14 Negative IM 6/ 50 Day 0 Day PBS / 2 × 600 μl control (PBS) 28 15 Positive control SC6 54 50 Day 0 / 1 × 1250 μl (VARIVAX ®) (pfu) 16 Positive control SC 654 50 Day 0 Day / (VARIVAX ®) (pfu) 28 17 Positive control SC 4 675 100Day 0 / 4 × 220 μl (VARIVAX ®) (pfu) 18 Positive control SC 4 675 100Day 0 Day / (VARIVAX ®) (pfu) 28

TABLE 5 Schedule of Sample Collection Pre- Day0 + Day Day28 + Day Day G#Antigen bleed 6 h 14 Day27 6 h 42 56 1 VZV-gE-oka- √ √ √ √ √ √ hIgkappa(G5; cap1) 2 VZV-gE-oka- √ √ √ √ √ √ √ hIgkappa (G5; cap1) 3 VZV-gE-oka-√ √ √ √ √ √ hIgkappa (G5; cap1) 4 VZV-gE-oka- √ √ √ √ √ √ √ hIgkappa(G5; cap1) 5 VZV-gE-oka √ √ √ √ √ (G0; cap1) 6 VZV-gE-oka √ √ √ √ √ (G0;cap1) 7 VZV-gE-oka √ √ √ √ √ (G0; cap1) 8 VZV-gE-oka √ √ √ √ √ (G0;cap1) 9 VZV-gE-oka √ √ √ √ √ (G5; cap1) 10 VZV-gE-oka √ √ √ √ √ (G5;cap1) 11 VZV-gE-oka √ √ √ √ √ (G5; cap1) 12 VZV-gE-oka √ √ √ √ √ (G5;cap1) 13 PBS √ √ √ √ √ √ 14 PBS √ √ √ √ √ √ √ 15 Positive √ √ √ √ √control 16 Positive √ √ √ √ √ control 17 Positive √ √ √ √ √ control 18Positive √ √ √ √ √ control

Example 16: Immunogenicity Study—ELISA

The instant studies were designed to test the immunogenicity in BALB/Cmice of candidate VZV vaccines comprising a mRNA polynucleotide encodingglycoprotein gE from VZV. Mice were immunized with various VZV mRNAvaccine formulations at set intervals, and sera were collected aftereach immunization. The immunization schedule is provided in Table 2 ofExample 15. The sera collection schedule is set forth in Table 4 ofExample 15. Enzyme-linked immunosorbent assay (ELISA)

Serum antibody titers against VZV glycoprotein E were determined byEnzyme-linked immunosorbent assay (ELISA) using standard methods. In onestudy, the amount of anti-VZV gE mouse IgG was measured in the pre-bleedand in serum collected at day 14 and day 42 post vaccination in micevaccinated intramuscularly with two 10 μg doses of either: (1)VZV-gE-hIgkappa (SEQ ID NO: 93) having 5′ cap:m7G(5′)ppp(5′)G-2′-O-methyl and N1-methylpseudouridine chemicalmodification (#1 in Tables 2 and 3); (2) VZV-gE (SEQ ID NO: 135) having5′ cap: m7G(5′)ppp(5′)G-2′-O-methyl, and no chemical modification (#6 inTables 2 and 3) (SEQ ID NO: 135); (3) VZV-gE having 5′ cap:m7G(5′)ppp(5′)G-2′-O-methyl and N1-methylpseudouridine chemicalmodification (#10 in Tables 2 and 3); (4) VARIVAX® vaccine (positivecontrol); or (5) PBS (negative control).

FIGS. 5-7 show that there was a very strong immune response with allmRNA encoded VZV-gE vaccines tested relative to the current VARIVAX®vaccine. FIG. 5 shows that at day 14, the titer for anti-VZV-gE IgG wasabout 10 g/mL in the serum of mice vaccinated with vaccine candidate (1)VZV-gE-hIgkappa having 5′ cap: m7G(5′)ppp(5′)G-2′-O-methyl andN1-methylpseudouridine chemical modification and about 50 μg/mL in theserum of mice vaccinated with vaccine candidate (3) VZV-gE having 5′cap: m7G(5′)ppp(5′)G-2′-O-methyl and N1-methylpseudouridine chemicalmodification. The level of anti-VZV-gE IgG in the serum of micevaccinated with VARIVAX® was not detectable at day 14. At day 42, theamount of anti-VZV-gE IgG present in the serum of mice vaccinated withvaccine candidate (1) VZV-gE-hIgkappa having 5′ cap:m7G(5′)ppp(5′)G-2′-O-methyl and N1-methylpseudouridine chemicalmodification or vaccine candidate (3) VZV-gE having 5′ cap:m7G(5′)ppp(5′)G-2′-O-methyl and N1-methylpseudouridine chemicalmodification was almost 1000-fold greater than the amount of anti-VZV-gEIgG present in the serum of mice vaccinated with VARIVAX®. The amount ofanti-VZV gE IgG present in the serum of mice vaccinated with vaccinecandidate (2) VZV-gE having 5′ cap: m7G(5′)ppp(5′)G-2′-O-methyl and nochemical modification was almost 100-fold greater than the amount ofanti-VZV-gE IgG present in the serum of mice vaccinated with VARIVAX®.This study indicates that each of the VZV gE mRNA vaccines tested is amore immunogenic vaccine that the current VARIVAX® VZV vaccine.

Example 17: Immunogenicity Study—ELISA

The instant studies were designed to test the immunogenicity in BALB/Cmice of candidate VZV vaccines comprising a mRNA polynucleotide encodingglycoprotein gE from VZV. Mice were immunized with various VZV mRNAvaccine formulations at set intervals, and sera were collected aftereach immunization. The immunization schedule is provided in Table 4 ofExample 15. The sera collection schedule is set forth in Table 5 ofExample 15.

Serum antibody titers against VZV glycoprotein E was determined byEnzyme-linked immunosorbent assay (ELISA) using standard methods. In asecond expanded study, the serum samples were serially diluted to bringthe signal within the scope of detectability using ELISA. The amount ofanti-VZV gE mouse IgG was measured in serum collected at day 42 postvaccination in mice vaccinated intramuscularly with two 10 μg doses ofeither: (1) VZV-gE-hIgkappa having 5′ cap: m7G(5′)ppp(5′)G-2′-O-methyland N1-methylpseudouridine chemical modification (#1 in Tables 2 and 3);(2) VZV-gE having 5′ cap: m7G(5′)ppp(5′)G-2′-O-methyl and no chemicalmodification (#6 in Tables 2 and 3); (3) VZV-gE having 5′ cap:m7G(5′)ppp(5′)G-2′-O-methyl and N1-methylpseudouridine chemicalmodification (#10 in Tables 2 and 3); (4) VARIVAX® vaccine (positivecontrol); or (5) PBS (negative control). The concentration ofanti-VZV-gE mouse IgG was measured in 10-fold serial dilutions.

FIG. 6 shows that the strongest immune response was found in micevaccinated with vaccine candidate (1) VZV-gE-hIgkappa having 5′ cap:m7G(5′)ppp(5′)G-2′-O-methyl and N1-methylpseudouridine chemicalmodification. The second strongest response was found in mice vaccinatedwith vaccine candidate (3) VZV-gE having 5′ cap:m7G(5′)ppp(5′)G-2′-O-methyl and N1-methylpseudouridine chemicalmodification. The third strongest response was found in mice vaccinatedwith vaccine candidate (2) VZV-gE having 5′ cap:m7G(5′)ppp(5′)G-2′-O-methyl and no chemical modification. All three VZVgE mRNA vaccines generated a significantly greater immune response thanVARIVAX® vaccine.

FIG. 7 shows the amount of anti-VZV-gE mouse IgG present in micevaccinated with vaccines (1)-(4) and (5) negative control at day 3, day14, and day 42 post-vaccination.

Example 18: Immunogenicity Study

The instant studies are designed to test the immunogenicity in BALB/Cmice of candidate VZV vaccines comprising a mRNA polynucleotide encodingvariant glycoprotein gE from VZV. Mice were immunized with various VZVmRNA vaccine formulations at set intervals, and sera were collectedafter each immunization at indicated time points. The immunizationschedule is provided in Table 6 below. The sera collection schedule isset forth in Table 7 below.

The amount of anti-VZV gE mouse IgG is measured in serum collected atthe times indicated in Table 7 post vaccination in mice vaccinatedintramuscularly with two 10 μg or 2 g doses of the indicated constructs.All mRNAs used have the 5′ cap: m7G(5′)ppp(5′)G-2′-O-methyl andN1-methylpseudouridine chemical modification. ZOSTAVAX® was used as apositive control and was injected into mice intramuscularly with twiceclinical dose of 19400 pfu SC. PBS was used as negative control.

Antibody titers against the VZV gE variant polypeptides in the sera ofmice immunized with VZV gE variant mRNA vaccines indicated in Table 6were determined by enzyme-linked immunosorbent assay (ELISA). To performthe ELISA, wells of a plate were coated with VZV gE antigen (Abcam:ab43050) in PBS. 100 μl of the VZV gE antigen at a concentration of 1,2, or 4 μg/ml were used for coating overnight at 4° C. The wells werethen washed with 300 μl of PBST (PBS with 0.05% tween) 3 times. The VZVgE-coated wells were blocked with 200 μl of blocking butter containing1% Blotto in PBS for 30 minutes at room temperature. Mice seracontaining anti-VZV gE antibodies were diluted 1:2000 and then subjectto 1:3 serial dilutions using PBST. The diluted sera were added to theVZV gE-coated wells and incubated for 1 hour at room temperature. Asecondary antibody, rabbit anti-mouse conjugated to horseradishperoxidase (HRP, Abcam: ab6728) was diluted 1:1000 in PBST and 100 μl ofthe secondary antibody containing solution was added to the wells andincubated for 45 minutes at room temperature. 100 μl of HRP substrates,KPL TMB, were added the wells and incubated for 3 minutes at roomtemperature before 100 μl of a stop solution (2M H₂SO₄) was added tostop the HRP reaction. Signals generated from the HRP substrates weremeasured at A450. The results were shown in FIGS. 11A, 11B, 12A, 12B, 13and Tables 8 and 9.

FIG. 11A shows that all gE variants induced much stronger immuneresponse than ZOSTAVAX® after the two 10 μg doses. FIG. 11B show thatall gE variants induced much stronger immune response than ZOSTAVAX®after the two 2 μg doses. With both dosages, the gE variantsGE-del_574_Y569A and GE-del-562-IgKappaSP induced the strongest immuneresponse and the antibody titer measured in the sera of mice immunizedwith this gE variant is over 10 times more than the antibody titermeasured in the sera of mice immunized with ZOSTAVAX®, indicating thatthe GE-del_574_Y569A and GE-del_562-IgKappaSP mRNAs are superior vaccinecandidates against VZV.

FIG. 12A shows the amount of antibodies in titrated sera collected frommice immunized twice with 10 μg of VZV gE mRNA variants described inTable 6. FIG. 12B shows the amount of antibodies in titrated seracollected from mice immunized twice with 2 g of VZV gE mRNA variantsdescribed in Table 6. When the sera were diluted more than 100 fold, theantibody titer is higher in VZV gE variants vaccinated mice sera than inZOSTAVAX® vaccinated mice sera, suggesting that the VZV gE mRNA variantsinduced much stronger immune response than ZOSTAVAX® in mice. All theVZV gE mRNA variants tested showed comparable ability in inducing immuneresponse in mice.

FIG. 13 is a graph showing the anti-VZV gE immune response induced bythe VZV gE variant mRNA vaccines compared to ZOSTAVAX®. The VZV gEvariant GE-delete_from_574-Y569A induced immune response in mice that isabout 1 log greater than ZOSTAVAX®.

Table 9 summarizes the reciprocal IgG titer (IC50) in the sera collectedfrom mice immunized with 10 μg or 2 μg of the respective VZV gE mRNAstwice. GE-delete_from_574-Y569A induced strong immune response witheither 10 μg or 2 μg dosages. The Geometric Mean Titer (GMT) was used toindicate the immunogenic potential of the VZV gE variant mRNA vaccines.GE-delete_from_574-Y569A showed the highest GMT value, indicating thatit is the most efficacious in inducing immune response against VZV gE.

TABLE 8 Summary of IC₅₀ of the different VZV constructs Reciprocal IgGtiter (IC50) Name 10 ug 2 ug GE-FULL_AEAADA 5741 6378 GE-FULL_AEAADA_10306 3556 &_Y582G GE-del-562 11672 6445 GE-del-562-laKappaSP 16490 7939GE-del_574 9031 4082 GE-del_574_Y569A 11704 7291 GE_Oka_hIgkappa 117086448 GE_Oka_hIgkappa 7045 3672 GE-full_AEAADA_ 4457 8242 GI-fullGE-del_574_Y569A NA PBS NA NA Zostavax 860 860 Assay controls plate 1plate 2 % CV std mean CV VZV_gE_Oka_hIgkappa 12808 11078 7.22 862.511940.5 0.07

TABLE 9 Reciprocal anti-gE IgG titer (IC50) measured by ELISA Name IC50GMT GE-full_AEAADA 1188.5 4291.4 31915.4 1261.8 30408.9 1000GE-full_AEAADA_&_Y582G 1150.8 3181.3 25351.3 1000 1000 11168.6GE-del-562 1000 6921.5 47752.9 12676.5 12912.2 2032.4GE-del-562-IgKappaSP 1000 13140.1 13122 51760.7 84918.1 6792 GE-del_57413091.8 13795.9 6760.8 14223.3 28774 GE-del_574_Y569A 20941.1 48865.28810.5 43351.1 68076.9 GE_Oka_hIgkappa 24266.1 6763.9 3026.9 13213 52362786.1 GE_Oka 27227 9078.2 30903 7638.4 1000 9594 Zostavax 6397.3 2228.41000 1000 2660.7 3228.5

Example 19: VZV In Vitro Neutralization Assay

A VZV in vitro neutralization assay was performed to evaluate theanti-VZV gE antibodies in neutralizing VZV. The anti-VZV gE antibodieswere obtained by collecting the sera of mice vaccinated with VZV gEvariant mRNA vaccines. Mice were vaccinated with VZV gE variant mRNAvaccines at dosages or 10 μg or 2 μg as described in Table 6 and serawere collected 2 weeks post 2^(nd) immunization.

To perform the assay, mice sera were diluted 1:5 and then subjected to1:2 serial dilutions. VZV virus were added to the sera andneutralization was allowed to continue for 1 hour at room temperature.ARPE-19 cells were seeded in 96-wells one day before and the virus/serummixtures were added to ARPE-19 cells at 50-100 pfu per well. The ARPE-19cells were fixed on the next day and VZV-specific staining wasperformed. The plates were scanned and analyzed. Results of the VZV invitro neutralization assay were summarized in Table 10. Values in Table10 are serum dilutions showing 50% reduction in well-area coverage byVZV virus plaques. No reduction in plaque number was observed. As shownin Table 10, one replicate of serum from mice immunized withGE-delete_from_574-Y569A variant mRNA vaccine was able to reducewell-area coverage by VZV virus plaques at 1:80 dilution.

TABLE 10 In vitro neutralization assay 10ug 20ug Antigen Replicate1Replicate2 Replicate1 Replicate2 SE-VZV-GE-full_with_AEAADA 20 10 10 10SE-VZV-GE-full_with_AEAADA_and_Y582G 40 20 10 SE-VZV-GE-delete-562 40 2020 10 SE-VZV-GE-delete-562-replacedSP-withIgKappa 40 40 20 40SE-VZV-GE_truncated-delete_from_574 80 40 40 20SE-VZV-GE-truncated-delete_from_574_-_Y569A 20 80 10 10KB_VZV_gE_Oka_hIgkappa 40 <20 20 10 KB_VZV_gE_Oka 160 10 10 10SE-VZV-Gl-full <10 <10 <20 <20 SE-VZV-GE-full_with_AEAADA +SE-VZV-Gl-full 20 10 — — SE-VZV-GE-truncated-delete from_574_ -_Y569A<10 <10 — — PBS <20 <20 Positive control 20 20

Example 20: Immunogenicity in Mice

Herpes zoster (HZ) or shingles is a debilitating disease characterizedby a vesicular rash, with the most common complication beingpost-herpetic neuralgia (PHN). PHN is a constant and severe pain thatdevelops after clearance of the cutaneous outbreak, and can last forseveral years, thereby contributing to the high morbidity of affectedindividuals. HZ is caused by reactivation of latent varicella-zostervirus (VZV) from the sensory ganglia. Immune responses generated duringprimary VZV infection (chickenpox) have been shown to prevent thereactivation of latent VZV. However, the incidence of HZ is stronglyassociated with advancing age. Several investigations have shown that Tcell-mediated immune responses decline with increasing age and duringimmunosuppression, resulting in reactivation of VZV. Nonetheless, thelevels of anti-VZV antibodies remain relatively stable with increasingage, demonstrating that the humoral immune response may not besufficient for the prevention of HZ. Several studies have reported theinduction of VZV-specific CD4⁺ and CD8⁺ T cells, with CD4⁺ T cellsdominating the memory response.

The approved vaccine ZOSTAVAX® demonstrates around 60-70% efficacy in50-60 years adults and declines with age. Recently a subunit adjuvantedvaccine (Shingrix: gE protein+ASO1B) was shown to have ˜90% efficacy inall age group of adults 50+. However, this vaccine demonstrated grade 3severe AE's in 10% of the vaccinated subjects. Shingrix demonstratedabout a log fold better T and B cell response after two doses toZOSTAVAX®. In the present studies mRNA immunization with a gE constructwas investigated for immunogenicity in mice and NHP.

The instant study was defined to test the immunogenicity in BALB/C miceof candidate VZV vaccines comprising a mRNA polynucleotide encodingglycoprotein gE from VZV. Mice were immunized with various VZV mRNAvaccine formulations as set forth below in Table 11. Groups 1 to 5 wereprimed with ZOSTAVAX® to mimic a primary Varicella exposure and group 6was primed with mRNA construct VZV-gE-del_574_Y569A. All groups (groups1-6) were boosted on day 28, as shown in Table 11. The animals were bledon days −3, 21, 38 and 42. Blood and spleens were collected forserological and T cell analysis on days 38 and 42. As shown in FIG. 14A,all groups gave comparable anti-gE antibody responses to mRNAvaccination with the animal receiving mRNA for prime and boost (group 6)trending higher. As shown in FIG. 15A, all groups demonstrated CD4T-cell responses with mRNA prime and boost (group 6) trending towards ahigher response. As shown in FIG. 15B, variable CD8 T-cell responseswere noted with the mRNA prime and boost (group 6) demonstrating highestCD8 T-cell frequencies.

TABLE 11 Injection Schedule Primary Localization Number ImmunizationBoost (VERO/MeWo) mice (Day 0) (Day 28) of Boost per G# 10 μg 10 μgConstruct group 1 ZOSTAVAX ® VZV-gE-del_ Golgi (high) 10 574_Y569A &cell membrane (high) 2 ZOSTAVAX ® VZV-gE-Oka- Golgi (low) & 10 hIgKappacytoplasmic 3 ZOSTAVAX ® VZV-gE-Oka Golgi/Golgi 10 4 ZOSTAVAX ® VZV-gEfull_ Organelles & 10 with_AEAADA cytoplasmic/cell (SEQ ID NO: 58)_membrane and_Y582G 5 ZOSTAVAX ® VZV-gE-full with Golgi & diffuse 10AEAADA perinuclear/cell (SEQ ID NO: 58) membrane 6 VZV-gE- VZV-gE-del_Golgi (high) & cell 10 del_574_Y569A 574_Y569A membrane (high)

Example 21: Immunogenicity in Non-Human Primates

Based on the data in Example 20, the mRNA construct VZV-gE-del_574_Y569Awas further evaluated for immunogenicity in non-human primates. Threegroups of Rhesus monkeys were primed with mRNA (VZV-gE-del_574_Y569A) orZOSTAVAX® and boosted as set forth below in Table 12. The animals werebled at days 0, 14, 28, and 42 for serological and T-cell analysis.T-cell analysis was performed on days 0, 28, and 42. As shown in FIGS.16A and 16B, the mRNA prime and boost (group 1) gave the highest anti-gEtiters which were followed by ZOSTAVAX® prime, mRNA boost (group 2). Thelatter group (group 2) anti-gE titers were approximately 10× better thanthe ZOSTAVAX® prime, ZOSTAVAX® boost (group 3). As shown in FIGS. 16Cand 16D, no CD4-T cells producing IFNγ, IL-2 or TNFα were detected inthe ZOSTAVAX® prime, ZOSTAVAX® boost group (group 3). In contrast, asshown in FIGS. 16C and 16D, reasonable frequency of CD4 T-cellsproducing IFNγ, IL-2 or TNFα were detected in the mRNA prime, mRNA boostgroup (group 1) and the ZOSTAVAX® prime, mRNA boost group (group 2) andwere statistically undifferentiated. These data indicate that one doseof mRNA vaccination after ZOSTAVAX® exposure was equivalent to two dosesof mRNA vaccination in inducing comparable T-cell responses.

TABLE 12 Injection Schedule Primary Boost Number Rhesus Immunization(Day 28) macaques (male and G# (Day 0) 10 μg 10 μg female) per group 1VZV-gE- VZV-gE- 5 del_574_Y569A del_574_Y569A 2 ZOSTAVAX ® VZV-gE- 5del_574_Y569A 3 ZOSTAVAX ® ZOSTAVAX ® 5

Example 22: Expression and In Vivo Localization of VZV-gE-Del_574_Y569AVariants

Variants of mRNAs encoding the VZV-gE-del_574_Y569A (variant 7, SEQ IDNO: 107, ORF sequence: SEQ ID NO: 86) or VZV-gE-full length (SEQ ID NO:135) were constructed, where the nucleotide sequence of thehomopolymeric stretches (e.g., 6, 5, or 4 consecutive As) were altered(without altering the amino acid sequence) to reduce the frequency ofpotential indel formation. Next generation sequencing showed the absenceof significant indels in VZV-gE-del_574_Y569A variant 7(VZV-gE-del_574_Y569A-v7) (FIG. 17). Mass spectrometry detected very lowlevels of indels in a 4A stretch in nucleotides 760-771 of the ORF.

Next, the expression levels of VZV-gE-del_574_Y569A-v7 were tested inMewo and HeLa cells and compared to the VZV-gE-del_574_Y569A mRNAconstruct. The mRNA constructs (200 ng) were transfected into Mewo orHeLa cells, using the Mirus TF transfection reagent in 96-well plates.NTIX control mRNA and PBS control were used in 30 K of MeWo cells. Allcells were serially diluted (2 fold) 6 hours post transfection,permeabilized, stained, and analyzed by flow cytometry for FITC+ cells(cells with antigen expression). Results show that theVZV-gE-del_574_Y569A-v7 mRNA construct was expressed at a levelcomparable to the VZV-gE-del_574_Y569A mRNA construct in both Mewo cells(FIG. 18A) and HeLa cells (FIG. 18B).

Protein was then detected in cell lysates of HeLa cells transfected(lipoplexed with 3 μl LF2000 per well in a 24-well plate) with eitherthe VZV-gE-del_574_Y569A mRNA construct (500 ng) or theVZV-gE-del_574_Y569A-v7 mRNA construct (500 ng), serially diluted to 100μg or 20 μg. Western blots for the VZV-gE antigen 16 hours posttransfection showed that the VZV-gE-del_574_Y569A-v7 mRNA construct wasexpressed at a level comparable to the VZV-gE-del_574_Y569A mRNAconstruct in HeLa cells (FIGS. 19A and 19B).

Next, the in vivo trafficking and localization of different VZV gEantigens were assessed. HeLa or MeWo cells were plated on 96 well platesand transfected with mRNA constructs encoding NTFIX control, full lengthVZV-gE, VZV-gE-del_574_Y569A, or VZV-gE-del_574_Y569A-v7 withLipofectamine 2000 (100 ng mRNA/0.4 μl LF per well). Twenty-four hoursafter transfection, the cells were fixed in 4% PFA, permeabilized with0.5% TX-100, and co-stained with rabbit anti-GM130 (CST D6B1-Golgimarker) and mouse anti-gE (Abcam 52549) antibodies at room temperaturefor 1 h in blocking buffer (1% BSA in PBS). The samples were then washedto remove the primary antibody and were incubated with anti-rabbit Alexa647 and anti-mouse Alexa 488 conjugated antibodies for 30 minutes atroom temperature, followed by counterstaining with DAPI for nuclearlocalization and HCS blue mask for cell segmentation. Cells were imagedwith the Opera Phenix high throughput spinning disk confocal.

The expression level and the localization of each antigen werequantified. The antigens expressed at comparable levels (FIG. 20A) butVZV-gE-del_574_Y569A encoded by the VZV-gE-del_574_Y569A mRNA constructor the VZV-gE-del_574_Y569A-v7 mRNA construct showed lower golgilocalization compared to the full-length VZV-gE (FIG. 20B). A shift inthe localization of VZV-gE-del_574_Y569A (encoded by either theVZV-gE-del_574_Y569A mRNA construct or the VZV-gE-del_574_Y569A-v7 mRNAconstruct) from golgi to the cell membrane, compared to the full-lengthVZV gE, was observed in HeLA cells (FIG. 21A) and in MeWo cells (FIGS.21B and 21C).

TABLE 13 Exemplary VZV gE mRNA constructs mRNA ConstructORF of mRNA Construct (excluding the stop codon)SE_VZV_gE_full_indel_fixed AUGGGGACAGUUAAUAAACCUGUGGUGGGGGUAUUGAUGGGGUUCGGAAUUAUCACGGGAACGUUGCGUAUAACGAAUCCGGUCAGAGCAUCCGUCUUGCGAUACGAUGAUUUUCACAUCGAUGAAGACAAACUGGAUACAAACUCCGUAUAUGAGCCUUACUACCAUUCAGAUCAUGCGGAGUCUUCAUGGGUAAAUCGGGGAGAGUCUUCGCGAAAAGCGUACGAUCAUAACUCACCUUAUAUAUGGCCACGUAAUGAUUAUGAUGGAUUUUUAGAGAACGCACACGAACACCAUGGGGUGUAUAAUCAGGGCCGUGGUAUCGAUAGCGGGGAACGGUUAAUGCAACCCACACAAAUGUCUGCACAGGAGGAUCUUGGGGACGAUACGGGCAUCCACGUUAUCCCUACGUUAAACGGCGAUGACAGACAUAAAAUUGUAAAUGUGGACCAACGUCAAUACGGUGACGUGUUUAAAGGAGAUCUUAAUCCAAAACCCCAAGGCCAAAGACUCAUUGAGGUGUCAGUGGAAGAAAAUCACCCGUUUACUUUACGCGCACCGAUUCAGCGGAUUUAUGGAGUCCGGUACACCGAGACUUGGAGCUUUUUGCCGUCAUUAACCUGUACGGGAGACGCAGCGCCCGCCAUCCAGCAUAUAUGUUUAAAGCAUACAACAUGCUUUCAAGACGUGGUGGUGGAUGUGGAUUGCGCGGAAAAUACUAAAGAGGAUCAGUUGGCCGAAAUCAGUUACCGUUUUCAAGGUAAGAAGGAAGCGGACCAACCGUGGAUUGUUGUAAACACGAGCACACUGUUUGAUGAACUCGAAUUAGACCCACCCGAGAUUGAACCGGGUGUCUUGAAAGUACUUCGGACAGAGAAACAAUACUUGGGUGUGUACAUUUGGAACAUGCGCGGCUCCGAUGGUACGUCUACCUACGCCACGUUUUUGGUCACCUGGAAAGGGGAUGAGAAGACAAGAAACCCUACGCCCGCAGUAACUCCUCAACCAAGAGGGGCUGAGUUUCAUAUGUGGAAUUACCACUCGCAUGUAUUUUCAGUUGGUGAUACGUUUAGCUUGGCAAUGCAUCUUCAGUAUAAGAUACAUGAAGCGCCAUUUGAUUUGCUGUUAGAGUGGUUGUAUGUCCCCAUCGAUCCUACAUGUCAACCAAUGCGGUUAUAUUCUACGUGUUUGUAUCAUCCCAACGCACCCCAAUGCCUCUCUCAUAUGAAUUCCGGUUGUACAUUUACCUCGCCACAUUUAGCCCAGCGUGUUGCAAGCACAGUGUAUCAGAAUUGUGAACAUGCAGAUAACUACACCGCAUAUUGUCUGGGAAUAUCUCAUAUGGAGCCUAGCUUUGGUCUAAUCUUACACGACGGGGGCACCACGUUAAAGUUUGUAGAUACACCCGAGAGUUUGUCGGGAUUAUACGUUUUUGUGGUGUAUUUUAACGGGCAUGUUGAAGCCGUAGCAUACACUGUUGUAUCCACAGUAGAUCAUUUUGUAAACGCAAUUGAAGAGCGUGGAUUUCCGCCAACGGCCGGUCAGCCACCGGCGACUACUAAACCCAAGGAAAUUACCCCCGUAAACCCCGGAACGUCACCACUUCUACGAUAUGCCGCAUGGACCGGAGGGCUUGCAGCAGUAGUACUUUUAUGUCUCGUAAUAUUUUUAAUCUGUACGGCUAAACGAAUGAGGGUUAAAGCCUACAGGGUAGACAAGUCUCCUUACAAUCAGUCAAUGUACUAUGCAGGACUCCCUGUUGACGAUUUCGAAGACUCAGAGAGUACAGACACAGAAGAAGAAUUCGGAAACGCUAUAGGUGGCUCUCACGGAGGUAGCUCGUAUACAGUGUACAUCGAUAAAACCAGA (SEQ ID NO: 142) SE-VZV-GE-574-Y569A-v2AUGGGGACAGUUAAUAAACCUGUGGUGGGGGUAUUGAUGGGGUUCGGAAUUAUCACGGGAACGUUGCGUAUAACGAAUCCGGUCAGAGCAUCCGUCUUGCGAUACGAUGAUUUUCACAUCGAUGAAGACAAACUGGAUACAAACUCCGUAUAUGAGCCUUACUACCAUUCAGAUCAUGCGGAGUCUUCAUGGGUAAAUCGGGGAGAGUCUUCGCGAAAAGCGUACGAUCAUAACUCACCUUAUAUAUGGCCACGUAAUGAUUAUGAUGGAUUUUUAGAGAACGCACACGAACACCAUGGGGUGUAUAAUCAGGGCCGUGGUAUCGAUAGCGGGGAACGGUUAAUGCAACCCACACAAAUGUCUGCACAGGAGGAUCUUGGGGACGAUACGGGCAUCCACGUUAUCCCUACGUUAAACGGCGAUGACAGACAUAAAAUUGUAAAUGUGGACCAACGUCAAUACGGUGACGUGUUUAAAGGAGAUCUUAAUCCAAAACCCCAAGGCCAAAGACUCAUUGAGGUGUCAGUGGAAGAAAAUCACCCGUUUACUUUACGCGCACCGAUUCAGCGGAUUUAUGGAGUCCGGUACACCGAGACUUGGAGCUUUUUGCCGUCAUUAACCUGUACGGGAGACGCAGCGCCCGCCAUCCAGCAUAUAUGUUUAAAACAUACAACAUGCUUUCAAGACGUGGUGGUGGAUGUGGAUUGCGCGGAAAAUACUAAAGAGGAUCAGUUGGCCGAAAUCAGUUACCGUUUUCAAGGUAAGAAGGAAGCGGACCAACCGUGGAUUGUUGUAAACACGAGCACACUGUUUGAUGAACUCGAAUUAGACCCCCCCGAGAUUGAACCGGGUGUCUUGAAAGUACUUCGGACAGAGAAACAAUACUUGGGUGUGUACAUUUGGAACAUGCGCGGCUCCGAUGGUACGUCUACCUACGCCACGUUUUUGGUCACCUGGAAAGGGGAUGAGAAGACAAGAAACCCUACGCCCGCAGUAACUCCUCAACCAAGAGGGGCUGAGUUUCAUAUGUGGAAUUACCACUCGCAUGUAUUUUCAGUUGGUGAUACGUUUAGCUUGGCAAUGCAUCUUCAGUAUAAGAUACAUGAAGCGCCAUUUGAUUUGCUGUUAGAGUGGUUGUAUGUCCCCAUCGAUCCUACAUGUCAACCAAUGCGGUUAUAUUCUACGUGUUUGUAUCAUCCCAACGCACCCCAAUGCCUCUCUCAUAUGAAUUCCGGUUGUACAUUUACCUCGCCACAUUUAGCCCAGCGUGUUGCAAGCACAGUGUAUCAAAAUUGUGAACAUGCAGAUAACUACACCGCAUAUUGUCUGGGAAUAUCUCAUAUGGAGCCUAGCUUUGGUCUAAUCUUACACGACGGGGGCACCACGUUAAAGUUUGUAGAUACACCCGAGAGUUUGUCGGGAUUAUACGUUUUUGUGGUGUAUUUUAACGGGCAUGUUGAAGCCGUAGCAUACACUGUUGUAUCCACAGUAGAUCAUUUUGUAAACGCAAUUGAAGAGCGUGGAUUUCCGCCAACGGCCGGUCAGCCACCGGCGACUACUAAACCCAAGGAAAUUACCCCCGUAAACCCCGGAACGUCACCACUUCUACGAUAUGCCGCAUGGACCGGAGGGCUUGCAGCAGUAGUACUUUUAUGUCUCGUAAUAUUUUUAAUCUGUACGGCUAAACGAAUGAGGGUUAAAGCCGCCAGGGUAGACAAG (SEQ ID NO: 143)SE-VZV-GE-574-Y569A-v3 AUGGGGACAGUUAAUAAACCUGUGGUGGGGGUAUUGAUGGGGUUCGGAAUUAUCACGGGAACGUUGCGUAUAACGAAUCCGGUCAGAGCAUCCGUCUUGCGAUACGAUGAUUUUCACAUCGAUGAAGACAAACUGGAUACAAACUCCGUAUAUGAGCCUUACUACCAUUCAGAUCAUGCGGAGUCUUCAUGGGUAAAUCGGGGAGAGUCUUCGCGAAAAGCGUACGAUCAUAACUCACCUUAUAUAUGGCCACGUAAUGAUUAUGAUGGAUUUUUAGAGAACGCACACGAACACCAUGGGGUGUAUAAUCAGGGCCGUGGUAUCGAUAGCGGGGAACGGUUAAUGCAACCCACACAAAUGUCUGCACAGGAGGAUCUUGGGGACGAUACGGGCAUCCACGUUAUCCCUACGUUAAACGGCGAUGACAGACAUAAAAUUGUAAAUGUGGACCAACGUCAAUACGGUGACGUGUUUAAAGGAGAUCUUAAUCCAAAACCCCAAGGCCAAAGACUCAUUGAGGUGUCAGUGGAAGAAAAUCACCCGUUUACUUUACGCGCACCGAUUCAGCGGAUUUAUGGAGUCCGGUACACCGAGACUUGGAGCUUUUUGCCGUCAUUAACCUGUACGGGAGACGCAGCGCCCGCCAUCCAGCAUAUAUGUUUAAAACAUACAACAUGCUUUCAAGACGUGGUGGUGGAUGUGGAUUGCGCGGAAAAUACUAAAGAGGAUCAGUUGGCCGAAAUCAGUUACCGUUUUCAAGGUAAGAAGGAAGCGGACCAACCGUGGAUUGUUGUAAACACGAGCACACUGUUUGAUGAACUCGAAUUAGACCCACCCGAGAUUGAACCGGGUGUCUUGAAAGUACUUCGGACAGAGAAACAAUACUUGGGUGUGUACAUUUGGAACAUGCGCGGCUCCGAUGGUACGUCUACCUACGCCACGUUUUUGGUCACCUGGAAAGGGGAUGAGAAGACAAGAAACCCUACGCCCGCAGUAACUCCUCAACCAAGAGGGGCUGAGUUUCAUAUGUGGAAUUACCACUCGCAUGUAUUUUCAGUUGGUGAUACGUUUAGCUUGGCAAUGCAUCUUCAGUAUAAGAUACAUGAAGCGCCAUUUGAUUUGCUGUUAGAGUGGUUGUAUGUCCCCAUCGAUCCUACAUGUCAACCAAUGCGGUUAUAUUCUACGUGUUUGUAUCAUCCCAACGCACCCCAAUGCCUCUCUCAUAUGAAUUCCGGUUGUACAUUUACCUCGCCACAUUUAGCCCAGCGUGUUGCAAGCACAGUGUAUCAAAAUUGUGAACAUGCAGAUAACUACACCGCAUAUUGUCUGGGAAUAUCUCAUAUGGAGCCUAGCUUUGGUCUAAUCUUACACGACGGGGGCACCACGUUAAAGUUUGUAGAUACACCCGAGAGUUUGUCGGGAUUAUACGUUUUUGUGGUGUAUUUUAACGGGCAUGUUGAAGCCGUAGCAUACACUGUUGUAUCCACAGUAGAUCAUUUUGUAAACGCAAUUGAAGAGCGUGGAUUUCCGCCAACGGCCGGUCAGCCACCGGCGACUACUAAACCCAAGGAAAUUACCCCCGUAAACCCCGGAACGUCACCACUUCUACGAUAUGCCGCAUGGACCGGAGGGCUUGCAGCAGUAGUACUUUUAUGUCUCGUAAUAUUUUUAAUCUGUACGGCUAAACGAAUGAGGGUUAAAGCCGCCAGGGUAGACAAG (SEQ ID NO: 144)SE-VZV-GE-574-Y569A-v4 AUGGGGACAGUUAAUAAACCUGUGGUGGGGGUAUUGAUGGGGUUCGGAAUUAUCACGGGAACGUUGCGUAUAACGAAUCCGGUCAGAGCAUCCGUCUUGCGAUACGAUGAUUUUCACAUCGAUGAAGACAAACUGGAUACAAACUCCGUAUAUGAGCCUUACUACCAUUCAGAUCAUGCGGAGUCUUCAUGGGUAAAUCGGGGAGAGUCUUCGCGAAAGGCGUACGAUCAUAACUCACCUUAUAUAUGGCCACGUAAUGAUUAUGAUGGAUUUUUAGAGAACGCACACGAACACCAUGGGGUGUAUAAUCAGGGCCGUGGUAUCGAUAGCGGGGAACGGUUAAUGCAACCCACACAAAUGUCUGCACAGGAGGAUCUUGGGGACGAUACGGGCAUCCACGUUAUCCCUACGUUAAACGGCGAUGACAGACAUAAGAUUGUAAAUGUGGACCAACGUCAAUACGGUGACGUGUUUAAAGGAGAUCUUAAUCCAAAGCCCCAAGGCCAAAGACUCAUUGAGGUGUCAGUGGAAGAGAAUCACCCGUUUACUUUACGCGCACCGAUUCAGCGGAUUUAUGGAGUCCGGUACACCGAGACUUGGAGCUUUUUGCCGUCAUUAACCUGUACGGGAGACGCAGCGCCCGCCAUCCAGCAUAUAUGUUUAAAGCAUACAACAUGCUUUCAAGACGUGGUGGUGGAUGUGGAUUGCGCGGAGAAUACUAAAGAGGAUCAGUUGGCCGAAAUCAGUUACCGUUUUCAAGGUAAGAAGGAAGCGGACCAACCGUGGAUUGUUGUAAACACGAGCACACUGUUUGAUGAACUCGAAUUAGACCCCCCCGAGAUUGAACCGGGUGUCUUGAAAGUACUUCGGACAGAGAAACAAUACUUGGGUGUGUACAUUUGGAACAUGCGCGGCUCCGAUGGUACGUCUACCUACGCCACGUUUUUGGUCACCUGGAAAGGGGAUGAGAAGACAAGAAACCCUACGCCCGCAGUAACUCCUCAACCAAGAGGGGCUGAGUUUCAUAUGUGGAAUUACCACUCGCAUGUAUUUUCAGUUGGUGAUACGUUUAGCUUGGCAAUGCAUCUUCAGUAUAAGAUACAUGAAGCGCCAUUUGAUUUGCUGUUAGAGUGGUUGUAUGUCCCCAUCGAUCCUACAUGUCAACCAAUGCGGUUAUAUUCUACGUGUUUGUAUCAUCCCAACGCACCCCAAUGCCUCUCUCAUAUGAAUUCCGGUUGUACAUUUACCUCGCCACAUUUAGCCCAGCGUGUUGCAAGCACAGUGUAUCAGAAUUGUGAACAUGCAGAUAACUACACCGCAUAUUGUCUGGGAAUAUCUCAUAUGGAGCCUAGCUUUGGUCUAAUCUUACACGACGGGGGCACCACGUUAAAGUUUGUAGAUACACCCGAGAGUUUGUCGGGAUUAUACGUUUUUGUGGUGUAUUUUAACGGGCAUGUUGAAGCCGUAGCAUACACUGUUGUAUCCACAGUAGAUCAUUUUGUAAACGCAAUUGAAGAGCGUGGAUUUCCGCCAACGGCCGGUCAGCCACCGGCGACUACUAAACCCAAGGAAAUUACCCCCGUAAACCCCGGAACGUCACCACUUCUACGAUAUGCCGCAUGGACCGGAGGGCUUGCAGCAGUAGUACUUUUAUGUCUCGUAAUAUUUUUAAUCUGUACGGCUAAACGAAUGAGGGUUAAAGCCGCCAGGGUAGACAA (SEQ ID NO: 145)SE-VZV-GE-574-Y569A-v5 AUGGGGACAGUUAAUAAACCUGUGGUGGGGGUAUUGAUGGGGUUCGGAAUUAUCACGGGAACGUUGCGUAUAACGAAUCCGGUCAGAGCAUCCGUCUUGCGAUACGAUGAUUUUCACAUCGAUGAAGACAAACUGGAUACAAACUCCGUAUAUGAGCCUUACUACCAUUCAGAUCAUGCGGAGUCUUCAUGGGUAAAUCGGGGAGAGUCUUCGCGAAAGGCGUACGAUCAUAACUCACCUUAUAUAUGGCCACGUAAUGAUUAUGAUGGAUUUUUAGAGAACGCACACGAACACCAUGGGGUGUAUAAUCAGGGCCGUGGUAUCGAUAGCGGGGAACGGUUAAUGCAACCCACACAAAUGUCUGCACAGGAGGAUCUUGGGGACGAUACGGGCAUCCACGUUAUCCCUACGUUAAACGGCGAUGACAGACAUAAGAUUGUAAAUGUGGACCAACGUCAAUACGGUGACGUGUUUAAAGGAGAUCUUAAUCCAAAGCCCCAAGGCCAAAGACUCAUUGAGGUGUCAGUGGAAGAGAAUCACCCGUUUACUUUACGCGCACCGAUUCAGCGGAUUUAUGGAGUCCGGUACACCGAGACUUGGAGCUUUUUGCCGUCAUUAACCUGUACGGGAGACGCAGCGCCCGCCAUCCAGCAUAUAUGUUUAAAGCAUACAACAUGCUUUCAAGACGUGGUGGUGGAUGUGGAUUGCGCGGAGAAUACUAAAGAGGAUCAGUUGGCCGAAAUCAGUUACCGUUUUCAAGGUAAGAAGGAAGCGGACCAACCGUGGAUUGUUGUAAACACGAGCACACUGUUUGAUGAACUCGAAUUAGACCCACCCGAGAUUGAACCGGGUGUCUUGAAAGUACUUCGGACAGAGAAACAAUACUUGGGUGUGUACAUUUGGAACAUGCGCGGCUCCGAUGGUACGUCUACCUACGCCACGUUUUUGGUCACCUGGAAAGGGGAUGAGAAGACAAGAAACCCUACGCCCGCAGUAACUCCUCAACCAAGAGGGGCUGAGUUUCAUAUGUGGAAUUACCACUCGCAUGUAUUUUCAGUUGGUGAUACGUUUAGCUUGGCAAUGCAUCUUCAGUAUAAGAUACAUGAAGCGCCAUUUGAUUUGCUGUUAGAGUGGUUGUAUGUCCCCAUCGAUCCUACAUGUCAACCAAUGCGGUUAUAUUCUACGUGUUUGUAUCAUCCCAACGCACCCCAAUGCCUCUCUCAUAUGAAUUCCGGUUGUACAUUUACCUCGCCACAUUUAGCCCAGCGUGUUGCAAGCACAGUGUAUCAGAAUUGUGAACAUGCAGAUAACUACACCGCAUAUUGUCUGGGAAUAUCUCAUAUGGAGCCUAGCUUUGGUCUAAUCUUACACGACGGGGGCACCACGUUAAAGUUUGUAGAUACACCCGAGAGUUUGUCGGGAUUAUACGUUUUUGUGGUGUAUUUUAACGGGCAUGUUGAAGCCGUAGCAUACACUGUUGUAUCCACAGUAGAUCAUUUUGUAAACGCAAUUGAAGAGCGUGGAUUUCCGCCAACGGCCGGUCAGCCACCGGCGACUACUAAACCCAAGGAAAUUACCCCCGUAAACCCCGGAACGUCACCACUUCUACGAUAUGCCGCAUGGACCGGAGGGCUUGCAGCAGUAGUACUUUUAUGUCUCGUAAUAUUUUUAAUCUGUACGGCUAAACGAAUGAGGGUUAAAGCCGCCAGGGUAGACAAG (SEQ ID NO: 146)SE-VZV-GE-574-Y569A-v6 AUGGGGACAGUUAAUAAACCUGUGGUGGGGGUAUUGAUGGGGUUCGGAAUUAUCACGGGAACGUUGCGUAUAACGAAUCCGGUCAGAGCAUCCGUCUUGCGAUACGAUGAUUUUCACAUCGAUGAAGACAAACUGGAUACAAACUCCGUAUAUGAGCCUUACUACCAUUCAGAUCAUGCGGAGUCUUCAUGGGUAAAUCGGGGAGAGUCUUCGCGAAAAGCGUACGAUCAUAACUCACCUUAUAUAUGGCCACGUAAUGAUUAUGAUGGAUUUUUAGAGAACGCACACGAACACCAUGGGGUGUAUAAUCAGGGCCGUGGUAUCGAUAGCGGGGAACGGUUAAUGCAACCCACACAAAUGUCUGCACAGGAGGAUCUUGGGGACGAUACGGGCAUCCACGUUAUCCCUACGUUAAACGGCGAUGACAGACAUAAAAUUGUAAAUGUGGACCAACGUCAAUACGGUGACGUGUUUAAAGGAGAUCUUAAUCCAAAACCCCAAGGCCAAAGACUCAUUGAGGUGUCAGUGGAAGAAAAUCACCCGUUUACUUUACGCGCACCGAUUCAGCGGAUUUAUGGAGUCCGGUACACCGAGACUUGGAGCUUUUUGCCGUCAUUAACCUGUACGGGAGACGCAGCGCCCGCCAUCCAGCAUAUAUGUUUAAAGCAUACAACAUGCUUUCAAGACGUGGUGGUGGAUGUGGAUUGCGCGGAAAAUACUAAAGAGGAUCAGUUGGCCGAAAUCAGUUACCGUUUUCAAGGUAAGAAGGAAGCGGACCAACCGUGGAUUGUUGUAAACACGAGCACACUGUUUGAUGAACUCGAAUUAGACCCCCCCGAGAUUGAACCGGGUGUCUUGAAAGUACUUCGGACAGAGAAACAAUACUUGGGUGUGUACAUUUGGAACAUGCGCGGCUCCGAUGGUACGUCUACCUACGCCACGUUUUUGGUCACCUGGAAAGGGGAUGAGAAGACAAGAAACCCUACGCCCGCAGUAACUCCUCAACCAAGAGGGGCUGAGUUUCAUAUGUGGAAUUACCACUCGCAUGUAUUUUCAGUUGGUGAUACGUUUAGCUUGGCAAUGCAUCUUCAGUAUAAGAUACAUGAAGCGCCAUUUGAUUUGCUGUUAGAGUGGUUGUAUGUCCCCAUCGAUCCUACAUGUCAACCAAUGCGGUUAUAUUCUACGUGUUUGUAUCAUCCCAACGCACCCCAAUGCCUCUCUCAUAUGAAUUCCGGUUGUACAUUUACCUCGCCACAUUUAGCCCAGCGUGUUGCAAGCACAGUGUAUCAGAAUUGUGAACAUGCAGAUAACUACACCGCAUAUUGUCUGGGAAUAUCUCAUAUGGAGCCUAGCUUUGGUCUAAUCUUACACGACGGGGGCACCACGUUAAAGUUUGUAGAUACACCCGAGAGUUUGUCGGGAUUAUACGUUUUUGUGGUGUAUUUUAACGGGCAUGUUGAAGCCGUAGCAUACACUGUUGUAUCCACAGUAGAUCAUUUUGUAAACGCAAUUGAAGAGCGUGGAUUUCCGCCAACGGCCGGUCAGCCACCGGCGACUACUAAACCCAAGGAAAUUACCCCCGUAAACCCCGGAACGUCACCACUUCUACGAUAUGCCGCAUGGACCGGAGGGCUUGCAGCAGUAGUACUUUUAUGUCUCGUAAUAUUUUUAAUCUGUACGGCUAAACGAAUGAGGGUUAAAGCCGCCAGGGUAGACAAG (SEQ ID NO: 147)SE-VZV-GE-574-Y569A-v7 AUGGGGACAGUUAAUAAACCUGUGGUGGGGGUAUUGAUGGGGUUCGGAAUUAUCACGGGAACGUUGCGUAUAACGAAUCCGGUCAGAGCAUCCGUCUUGCGAUACGAUGAUUUUCACAUCGAUGAAGACAAACUGGAUACAAACUCCGUAUAUGAGCCUUACUACCAUUCAGAUCAUGCGGAGUCUUCAUGGGUAAAUCGGGGAGAGUCUUCGCGAAAAGCGUACGAUCAUAACUCACCUUAUAUAUGGCCACGUAAUGAUUAUGAUGGAUUUUUAGAGAACGCACACGAACACCAUGGGGUGUAUAAUCAGGGCCGUGGUAUCGAUAGCGGGGAACGGUUAAUGCAACCCACACAAAUGUCUGCACAGGAGGAUCUUGGGGACGAUACGGGCAUCCACGUUAUCCCUACGUUAAACGGCGAUGACAGACAUAAAAUUGUAAAUGUGGACCAACGUCAAUACGGUGACGUGUUUAAAGGAGAUCUUAAUCCAAAACCCCAAGGCCAAAGACUCAUUGAGGUGUCAGUGGAAGAAAAUCACCCGUUUACUUUACGCGCACCGAUUCAGCGGAUUUAUGGAGUCCGGUACACCGAGACUUGGAGCUUUUUGCCGUCAUUAACCUGUACGGGAGACGCAGCGCCCGCCAUCCAGCAUAUAUGUUUAAAGCAUACAACAUGCUUUCAAGACGUGGUGGUGGAUGUGGAUUGCGCGGAAAAUACUAAAGAGGAUCAGUUGGCCGAAAUCAGUUACCGUUUUCAAGGUAAGAAGGAAGCGGACCAACCGUGGAUUGUUGUAAACACGAGCACACUGUUUGAUGAACUCGAAUUAGACCCACCCGAGAUUGAACCGGGUGUCUUGAAAGUACUUCGGACAGAGAAACAAUACUUGGGUGUGUACAUUUGGAACAUGCGCGGCUCCGAUGGUACGUCUACCUACGCCACGUUUUUGGUCACCUGGAAAGGGGAUGAGAAGACAAGAAACCCUACGCCCGCAGUAACUCCUCAACCAAGAGGGGCUGAGUUUCAUAUGUGGAAUUACCACUCGCAUGUAUUUUCAGUUGGUGAUACGUUUAGCUUGGCAAUGCAUCUUCAGUAUAAGAUACAUGAAGCGCCAUUUGAUUUGCUGUUAGAGUGGUUGUAUGUCCCCAUCGAUCCUACAUGUCAACCAAUGCGGUUAUAUUCUACGUGUUUGUAUCAUCCCAACGCACCCCAAUGCCUCUCUCAUAUGAAUUCCGGUUGUACAUUUACCUCGCCACAUUUAGCCCAGCGUGUUGCAAGCACAGUGUAUCAGAAUUGUGAACAUGCAGAUAACUACACCGCAUAUUGUCUGGGAAUAUCUCAUAUGGAGCCUAGCUUUGGUCUAAUCUUACACGACGGGGGCACCACGUUAAAGUUUGUAGAUACACCCGAGAGUUUGUCGGGAUUAUACGUUUUUGUGGUGUAUUUUAACGGGCAUGUUGAAGCCGUAGCAUACACUGUUGUAUCCACAGUAGAUCAUUUUGUAAACGCAAUUGAAGAGCGUGGAUUUCCGCCAACGGCCGGUCAGCCACCGGCGACUACUAAACCCAAGGAAAUUACCCCCGUAAACCCCGGAACGUCACCACUUCUACGAUAUGCCGCAUGGACCGGAGGGCUUGCAGCAGUAGUACUUUUAUGUCUCGUAAUAUUUUUAAUCUGUACGGCUAAACGAAUGAGGGUUAAAGCCGCCAGGGUAGACAAG (SEQ ID NO: 148)SE-VZV-GE-574-Y569A-v8 AUGGGGACAGUUAAUAAACCUGUGGUGGGCGUAUUGAUGGGGUUCGGAAUUAUCACGGGAACGUUGCGUAUAACGAAUCCGGUCAGAGCAUCCGUCUUGCGAUACGAUGAUUUUCACAUCGAUGAAGACAAACUGGAUACAAACUCCGUAUAUGAGCCUUACUACCAUUCAGAUCAUGCGGAGUCUUCAUGGGUAAAUCGGGGAGAGUCUUCGCGAAAGGCGUACGAUCAUAACUCACCUUAUAUAUGGCCACGUAAUGAUUAUGAUGGAUUCUUAGAGAACGCACACGAACACCAUGGGGUGUAUAAUCAGGGCCGUGGUAUCGAUAGCGGGGAACGGUUAAUGCAACCCACACAAAUGUCUGCACAGGAGGAUCUUGGGGACGAUACGGGCAUCCACGUUAUCCCUACGUUAAACGGCGAUGACAGACAUAAGAUUGUAAAUGUGGACCAACGUCAAUACGGUGACGUGUUUAAAGGAGAUCUUAAUCCAAAGCCCCAAGGCCAAAGACUCAUUGAGGUGUCAGUGGAAGAGAAUCACCCGUUUACUUUACGCGCACCGAUUCAGCGGAUUUAUGGAGUCCGGUACACCGAGACUUGGAGCUUCUUGCCGUCAUUAACCUGUACGGGAGACGCAGCGCCCGCCAUCCAGCAUAUAUGUUUAAAGCAUACAACAUGCUUUCAAGACGUGGUGGUGGAUGUGGAUUGCGCGGAGAAUACUAAAGAGGAUCAGUUGGCCGAAAUCAGUUACCGUUUUCAAGGUAAGAAGGAAGCGGACCAACCGUGGAUUGUUGUAAACACGAGCACACUGUUUGAUGAACUCGAAUUAGACCCACCCGAGAUUGAACCGGGUGUCUUGAAAGUACUUCGGACAGAGAAACAAUACUUGGGUGUGUACAUUUGGAACAUGCGCGGCUCCGAUGGUACGUCUACCUACGCCACGUUCUUGGUCACCUGGAAAGGGGAUGAGAAGACAAGAAACCCUACGCCCGCAGUAACUCCUCAACCAAGAGGGGCUGAGUUUCAUAUGUGGAAUUACCACUCGCAUGUAUUUUCAGUUGGUGAUACGUUUAGCUUGGCAAUGCAUCUUCAGUAUAAGAUACAUGAAGCGCCAUUUGAUUUGCUGUUAGAGUGGUUGUAUGUCCCCAUCGAUCCUACAUGUCAACCAAUGCGGUUAUAUUCUACGUGUUUGUAUCAUCCCAACGCACCCCAAUGCCUCUCUCAUAUGAAUUCCGGUUGUACAUUUACCUCGCCACAUUUAGCCCAGCGUGUUGCAAGCACAGUGUAUCAGAAUUGUGAACAUGCAGAUAACUACACCGCAUAUUGUCUGGGAAUAUCUCAUAUGGAGCCUAGCUUUGGUCUAAUCUUACACGACGGAGGCACCACGUUAAAGUUUGUAGAUACACCCGAGAGUUUGUCGGGAUUAUACGUCUUUGUGGUGUAUUUUAACGGGCAUGUUGAAGCCGUAGCAUACACUGUUGUAUCCACAGUAGAUCAUUUUGUAAACGCAAUUGAAGAGCGUGGAUUUCCGCCAACGGCCGGUCAGCCACCGGCGACUACUAAACCCAAGGAAAUUACGCCCGUAAACCCCGGAACGUCACCACUUCUACGAUAUGCCGCAUGGACCGGAGGGCUUGCAGCAGUAGUACUUUUAUGUCUCGUAAUAUUCUUAAUCUGUACGGCUAAACGAAUGAGGGUUAAAGCCGCCAGGGUAGACAAG (SEQ ID NO: 149)VZV-GE-Truncated-modified- AUGGGCACCGUGAACAAGCCUGUUGUGGGCGUGCUGAUGGGCUUV9 CGGCAUCAUCACAGGCACCCUGCGGAUCACCAAUCCUGUGCGGGCUAGCGUGCUGAGAUACGACGACUUCCACAUCGACGAGGACAAGCUGGACACCAACAGCGUGUACGAGCCCUACUACCACAGCGAUCACGCCGAGUCUAGCUGGGUCAACAGAGGCGAGAGCAGCAGAAAGGCCUACGACCACAACAGCCCUUACAUCUGGCCCAGAAACGACUACGACGGCUUCCUCGAGAAUGCCCACGAACACCACGGCGUGUACAAUCAAGGCAGAGGCAUCGACAGCGGCGAGAGACUGAUGCAGCCUACACAGAUGAGCGCCCAAGAGGACCUGGGAGAUGAUACCGGCAUCCACGUGAUCCCUACACUGAACGGCGACGACCGGCACAAGAUCGUGAACGUGGACCAGAGACAGUACGGCGACGUGUUCAAGGGCGACCUGAAUCCUAAGCCUCAGGGCCAGCGCCUGAUCGAGGUUUCCGUGGAAGAGAAUCACCCUUUCACACUGCGGGCUCCCAUCCAGAGAAUCUACGGCGUGCGCUAUACCGAGACAUGGUCCUUUCUGCCCAGCCUGACAUGUACCGGCGACGCCGCUCCUGCCAUCCAGCACAUUUGUCUGAAGCACACCACCUGUUUCCAGGACGUGGUGGUGGAUGUGGACUGCGCCGAGAACACCAAAGAGGAUCAGCUGGCCGAGAUCAGCUACCGGUUCCAGGGAAAGAAAGAGGCCGACCAGCCUUGGAUCGUGGUCAACACCAGCACACUGUUCGACGAGCUGGAACUGGACCCUCCUGAGAUUGAACCCGGCGUCCUGAAGGUGCUGAGAACCGAGAAGCAGUACCUGGGAGUGUACAUCUGGAACAUGAGAGGCAGCGACGGCACCUCUACCUACGCCACCUUUCUGGUCACAUGGAAGGGCGACGAGAAGACCAGAAAUCCCACACCAGCCGUGACACCUCAGCCUAGAGGCGCCGAAUUUCACAUGUGGAACUACCACUCUCACGUGUUCAGCGUGGGCGAUACCUUCAGCCUGGCCAUGCAUCUGCAGUACAAGAUCCACGAGGCUCCCUUCGACCUGCUGCUGGAAUGGCUGUACGUGCCCAUCGAUCCUACCUGCCAGCCUAUGCGGCUGUACUCCACCUGUCUGUAUCACCCUAACGCUCCUCAGUGCCUGAGCCACAUGAAUAGCGGCUGCACCUUCACAAGCCCUCACCUGGCUCAGCGAGUGGCCAGCACAGUGUACCAGAAUUGCGAGCACGCCGACAAUUACACCGCCUACUGUCUGGGCAUCAGCCACAUGGAACCUAGCUUCGGCCUGAUCCUGCACGAUGGCGGCACCACACUGAAGUUCGUGGACACACCUGAGAGCCUGAGCGGCCUGUAUGUGUUUGUGGUGUACUUCAACGGCCACGUGGAAGCCGUGGCCUACACCGUGGUGUCUACCGUGGACCACUUCGUGAACGCCAUCGAGGAAAGAGGCUUCCCUCCAACUGCUGGACAGCCUCCUGCCACCACCAAGCCUAAAGAAAUCACACCCGUGAAUCCCGGCACUAGCCCUCUGCUUAGAUACGCCGCUUGGACAGGCGGACUGGCUGCUGUUGUUCUGCUGUGCCUGGUCAUCUUCCUGAUCUGCACCGCCAAGCGGAUGAGAGUGAAAGCCGCCAGAGUGGACAAG (SEQ ID NO: 150) Corresponding amino acidMGTVNKPVVGVLMGFGIITGTLRITNPVRASVLRYDDFHIDEDKLDTN sequenceSVYEPYYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLGDDTGIHVIPTLNGDDRHKIVNVDQRQYGDVFKGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVLKVLRTEKQYLGVYIWNMRGSDGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNSGCTFTSPHLAQRVASTVYQNCEHADNYTAYCLGISHMEPSFGLILHDGGTTLKFVDTPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITPVNPGTSPLLRYAAWTGGLAAVVLLCLVIFLICTAKRMRVKAARVDK (SEQ ID NO: 38) 5′ UTRGGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAAGAGCC ACC (SEQ ID NO: 138) 3′ UTRUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGC (SEQ ID NO: 139) PolyA Tail100 nt (SEQ ID NO: 140)

TABLE 14 Varicella zoster virus Amino Acid Sequences GenBank ProteinName Accession glycoprotein B envelope glycoprotein B [Human herpesvirus3] NP_040154.2 glycoprotein B ORF31 [Human herpesvirus 3] AKG57704.1glycoprotein B ORF 31 [Human herpesvirus 3] AIT52967.1 glycoprotein Benvelope glycoprotein B [Human herpesvirus 3] AFJ68532.1 glycoprotein BORF31 [Human herpesvirus 3] AKG57414.1 glycoprotein B ORF31 [Humanherpesvirus 3] AKG58507.1 glycoprotein B RecName: Full = Envelopeglycoprotein B; Short = gB; Q4JR05.2 AltName: Full = Glycoprotein II;Flags: Precursor [Human herpesvirus 3 strain Oka vaccine] glycoprotein BORF31 [Human herpesvirus 3] AEL30845.1 glycoprotein B glycoprotein B[Human herpesvirus 3] AAK01041.1 glycoprotein B glycoprotein B [Humanherpesvirus 3] AEW89232.1 glycoprotein B glycoprotein B [Humanherpesvirus 3] AEW88728.1 glycoprotein B ORF31 [Human herpesvirus 3]AAK19938.1 glycoprotein B glycoprotein B [Human herpesvirus 3]AAP32845.1 glycoprotein B ORF 31 [Human herpesvirus 3] AHJ08729.1glycoprotein B ORF31 [Human herpesvirus 3] AAY57715.1 glycoprotein BORF31 [Human herpesvirus 3] AGY33726.1 glycoprotein B ORF 31 [Humanherpesvirus 3] AHJ09321.1 glycoprotein B ORF31 [Human herpesvirus 3]AAY57644.1 glycoprotein B ORF 31 [Human herpesvirus 3] AHJ09025.1glycoprotein B glycoprotein B [Human herpesvirus 3] AEW88584.1glycoprotein B ORF 31 [Human herpesvirus 3] AHJ09099.1 glycoprotein BORF31 [Human herpesvirus 3] AGY33060.1 glycoprotein B ORF 31 [Humanherpesvirus 3] AHJ09395.1 glycoprotein C RecName: Full = Envelopeglycoprotein C; Short = gC; P09256.2 AltName: Full = Glycoprotein V;Short = gpV glycoprotein C envelope glycoprotein gC [Human herpesvirus3] ABH08453.1 glycoprotein C ORF14 [Human herpesvirus 3] AIH07125.1glycoprotein C unknown protein [Human herpesvirus 3] AAA69563.1glycoprotein C ORF14 [Human herpesvirus 3] AIH07051.1 glycoprotein CORF14 [Human herpesvirus 3] AIJ28607.1 glycoprotein C ORF14 [Humanherpesvirus 3] AEL30828.1 glycoprotein C envelope glycoprotein gC [Humanherpesvirus 3] ABE03032.1 glycoprotein C envelope glycoprotein gC [Humanherpesvirus 3] ABE67122.1 glycoprotein C envelope glycoprotein C [Humanherpesvirus 3] NP_040137.1 glycoprotein C membrane glycoprotein C [Humanherpesvirus 3] AEW88351.1 glycoprotein C envelope glycoprotein C [Humanherpesvirus 3] AFJ68515.1 glycoprotein C membrane glycoprotein C [Humanherpesvirus 3] AAT07696.1 glycoprotein C envelope glycoprotein gC [Humanherpesvirus 3] ABF22098.1 glycoprotein C membrane glycoprotein C [Humanherpesvirus 3] AEW89287.1 glycoprotein C glycoprotein C [Humanherpesvirus 3] AGC94505.1 glycoprotein C envelope glycoprotein gC [Humanherpesvirus 3] ABF21514.1 glycoprotein C envelope glycoprotein gC [Humanherpesvirus 3] ABF21879.1 glycoprotein C envelope glycoprotein gC [Humanherpesvirus 3] ABF21587.1 glycoprotein C ORF 14 [Human herpesvirus 3]AIT53315.1 glycoprotein C membrane glycoprotein C [Human herpesvirus 3]AEW89215.1 glycoprotein C envelope glycoprotein gC [Human herpesvirus 3]ABF21660.1 glycoprotein C membrane glycoprotein C [Human herpesvirus 3]AEW88567.1 glycoprotein C envelope glycoprotein gC [Human herpesvirus 3]CAI44857.1 glycoprotein C envelope glycoprotein C [Human herpesvirus 3]AHB80244.1 glycoprotein C ORF14 [Human herpesvirus 3] AAY57702.1glycoprotein C glycoprotein c [Human herpesvirus 3] AGS32072.1glycoprotein C envelope glycoprotein gC [Human herpesvirus 3] AGL50971.1glycoprotein C membrane glycoprotein C [Human herpesvirus 3] AAT07772.1glycoprotein C membrane glycoprotein C [Human herpesvirus 3] AEW88495.1glycoprotein C ORF 14 [Human herpesvirus 3] AIT53461.1 glycoprotein CORF 14 [Human herpesvirus 3] AIT52950.1 glycoprotein C ORF14 [Humanherpesvirus 3] AAY57631.1 glycoprotein C envelope glycoprotein gC [Humanherpesvirus 3] ABF21952.1 glycoprotein C membrane glycoprotein C [Humanherpesvirus 3] AEW89143.1 glycoprotein C membrane glycoprotein C [Humanherpesvirus 3] AEW88783.1 glycoprotein C membrane glycoprotein C [Humanherpesvirus 3] AEW88999.1 glycoprotein C membrane glycoprotein C [Humanherpesvirus 3] AEW88063.1 glycoprotein C membrane glycoprotein C [Humanherpesvirus 3] AEW89071.1 glycoprotein C membrane glycoprotein C [Humanherpesvirus 3] AEW88639.1 glycoprotein C membrane glycoprotein C [Humanherpesvirus 3] AEW87991.1 glycoprotein C ORF 14 [Human herpesvirus 3]AIT53753.1 glycoprotein C ORF 14 [Human herpesvirus 3] AIT53096.1glycoprotein C envelope glycoprotein gC [Human herpesvirus 3] ABF22025.1glycoprotein C envelope glycoprotein gC [Human herpesvirus 3] AFO85518.1glycoprotein C envelope glycoprotein gC [Human herpesvirus 3] ABF21733.1glycoprotein C envelope glycoprotein gC [Human herpesvirus 3] ABF21806.1glycoprotein C membrane glycoprotein C [Human herpesvirus 3] AEW89359.1glycoprotein C membrane glycoprotein C [Human herpesvirus 3] AEW88855.1glycoprotein C envelope glycoprotein gC [Human herpesvirus 3] AFO85591.1glycoprotein C membrane glycoprotein C [Human herpesvirus 3] AEW89431.1glycoprotein C membrane glycoprotein C [Human herpesvirus 3] AEW88711.1glycoprotein C membrane glycoprotein C [Human herpesvirus 3] AEW88135.1glycoprotein C membrane glycoprotein C [Human herpesvirus 3] AEW88927.1glycoprotein C ORF14 [Human herpesvirus 3] AKG56156.1 glycoprotein CORF14 [Human herpesvirus 3] AKG57178.1 glycoprotein C ORF14 [Humanherpesvirus 3] AKG58125.1 glycoprotein C ORF14 [Human herpesvirus 3]AGY32970.1 glycoprotein C ORF14 [Human herpesvirus 3] AKG56229.1glycoprotein C ORF14 [Human herpesvirus 3] AGY32896.1 glycoprotein CORF14 [Human herpesvirus 3] AKG56521.1 glycoprotein C ORF 14 [Humanherpesvirus 3] AHJ08712.1 glycoprotein E unknown [Human herpesvirus 3]ABE03086.1 glycoprotein E glycoprotein E [Human herpesvirus 3]AAK01047.1 glycoprotein E RecName: Full = Envelope glycoprotein E; Short= gE; Q9J3M8.1 Flags: Precursor glycoprotein E membrane glycoprotein E[Human herpesvirus 3] AEW88548.1 glycoprotein E ORF68 [Human herpesvirus3] AGY33616.1 glycoprotein E membrane glycoprotein E [Human herpesvirus3] AEW89124.1 glycoprotein E ORF 68 [Human herpesvirus 3] AIT53150.1glycoprotein E unnamed protein product [Human herpesvirus 3] CAA25033.1glycoprotein E envelope glycoprotein E [Human herpesvirus 3] NP_040190.1glycoprotein E ORF68 [Human herpesvirus 3] AKG56356.1 glycoprotein Emembrane glycoprotein E [Human herpesvirus 3] AEW89412.1 glycoprotein Emembrane glycoprotein gE [Human herpesvirus 3] ABF21714.1 glycoprotein Emembrane glycoprotein E [Human herpesvirus 3] AAT07749.1 glycoprotein Emembrane glycoprotein E [Human herpesvirus 3] AEW88764.1 glycoprotein Eglycoprotein E [Human herpesvirus 3] AAG48520.1 glycoprotein E membraneglycoprotein E [Human herpesvirus 3] AEW88980.1 glycoprotein H envelopeglycoprotein H [Human herpesvirus 3] NP_040160.1 glycoprotein Hglycoprotein H [Human herpesvirus 3] AEW89454.1 glycoprotein H ORF37[Human herpesvirus 3 VZV-32] AAK19252.1 glycoprotein H RecName: Full =Envelope glycoprotein H; Short = gH; Q775J3.1 AltName: Full =Glycoprotein III; Short = GPIII; Flags:Precursor glycoprotein Hglycoprotein H [Human herpesvirus 3] AAK01042.1 glycoprotein H ORF37[Human herpesvirus 3] AKG58587.1 glycoprotein H ORF37 [Human herpesvirus3] AGY33215.1 glycoprotein H glycoprotein H [Human herpesvirus 3]AAP32857.1 glycoprotein H envelope glycoprotein gH [Human herpesvirus 3]ABE03056.1 glycoprotein H ORF 37 [Human herpesvirus 3] AHJ09328.1glycoprotein H glycoprotein H [Human herpesvirus 3] AAP32862.1glycoprotein H ORF37 [Human herpesvirus 3] AKG57421.1 glycoprotein HORF37 [Human herpesvirus 3] AKG56618.1 glycoprotein H ORF37 [Humanherpesvirus 3] AKG56545.1 glycoprotein H glycoprotein H [Humanherpesvirus 3] AEW89382.1 glycoprotein H glycoprotein H [Humanherpesvirus 3] AGC94548.1 glycoprotein I envelope glycoprotein I [Humanherpesvirus 3] NP_040189.1 glycoprotein I membrane glycoprotein I [Humanherpesvirus 3] AEW89195.1 glycoprotein I ORF67 [Human herpesvirus 3]AKG58616.1 glycoprotein I ORF67 [Human herpesvirus 3] AGY34059.1glycoprotein I membrane glycoprotein I [Human herpesvirus 3] AEW89051.1glycoprotein I ORF67 [Human herpesvirus 3 VZV-32] AAK19249.1glycoprotein I membrane glycoprotein I [Human herpesvirus 3] AEW89483.1glycoprotein K envelope glycoprotein K [Human herpesvirus 3] NP_040128.1glycoprotein K glycoprotein K [Human herpesvirus 3] AEW88773.1glycoprotein K ORF 5 [Human herpesvirus 3] AHJ09368.1 glycoprotein KORF5 [Human herpesvirus 3] AKG58699.1 glycoprotein K glycoprotein K[Human herpesvirus 3] AEW88701.1 glycoprotein K ORF5 [Human herpesvirus3] AKG56803.1 glycoprotein K glycoprotein K [Human herpesvirus 3]AEW88053.1 glycoprotein L RecName: Full = Envelope glycoprotein L; Short= gL; Q9J3N1.1 Flags: Precursor glycoprotein L virion glycoprotein gL[Human herpesvirus 3] ABE03078.1 glycoprotein L glycoprotein L [Humanherpesvirus 3] AGM33094.1 glycoprotein L ORF60 [Human herpesvirus 3]AKG56786.1 glycoprotein L envelope glycoprotein L [Human herpesvirus 3]NP_040182.1 glycoprotein L virion glycoprotein gL [Human herpesvirus 3]ABF21706.1 glycoprotein M envelope glycoprotein M [Human herpesvirus 3]NP_040172.1 glycoprotein M ORF 50 [Human herpesvirus 3] AIT53351.1glycoprotein M ORF50 [Human herpesvirus 3] AKG56119.1 glycoprotein MORF50 [Human herpesvirus 3] AGY33080.1 glycoprotein M envelopeglycoprotein gM [Human herpesvirus 3] ABE03068.1 glycoprotein M virionmembmne glycoprotein M [Human herpesvirus 3] AEW88530.1 glycoprotein Mvirion membmne glycoprotein M [Human herpesvirus 3] AEW88674.1glycoprotein N envelope glycoprotein N [Human herpesvirus 3] YP_068406.1glycoprotein N ORF9a [Human herpesvirus 3] AGY33038.1 glycoprotein Nmembrane protein [Human herpesvirus 3] AAT07690.1 glycoprotein Nmembrane protein [Human herpesvirus 3] AEW88273.1 glycoprotein Nmembrane protein [Human herpesvirus 3] AEW88489.1

TABLE 15 VZV Polypeptide Sequences Name Sequence SEQ ID NO:gi|443500676|gb| MGTVNKPVVGVLMGFGIITGTLRITNPVRASVLRYDDFH 45 AGC94542.1|IDEDKLDTNSVYEPYYHSDHAESSWVNRGESSRKAYDH glycoprotein ENSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLM [HumanQPTQMSAQEDLGDDTGIHVIPTLNGDDRHKIVNVDQRQ herpesvirus 3]YGDVFKGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVLKVLRTEKQYLGVYIWNMRGSDGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNSGCTFTSPHLAQRVASTVYQNCEHADNYTAYCLGISHMEPSFGLILHDGGTTLKFVDTPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITPVNPGTSPLLRYAAWTGGLAAVVLLCLVIFLICTAKRMRVKAYRVDKSPYNQSMYYAGLPVDDFEDSESTDTEEEFGNAIGGSHGGSSYTVY IDKTR gi|443500675|gb|MFLIQCLISAVIFYIQVTNALIFKGDHVSLQVNSSLTSILIP 46 AGC94541.1|MQNDNYTEIKGQLVFigEQLPTGTNYSGTLELLYADTVA glycoprotein IFCFRSVQVIRYDGCPRIRTSAFISCRYKHSWHYGNSTDRI [HumanSTEPDAGVMLKITKPGINDAGVYVLLVRLDHSRSTDGFI herpesvirus 3]LGVNVYTAGSHHNIHGVIYTSPSLQNGYSTRALFQQARLCDLPATPKGSGTSLFQHMLDLRAGKSLEDNPWLHEDVVTTETKSVVKEGIENHVYPTDMSTLPEKSLNDPPENLLIIIPIVASVMILTAMVIVIVISVKRRRIKKHPIYRPNTKTRRGIQNATPESDVMLEAAIAQLATIREESPPHSVVNPFVK VZV-GE-delete-MGTVNKPVVGVLMGFGIITGTLRITNPVRASVLRYDDFH 47 562IDEDKLDTNSVYEPYYHSDHAESSWVNRGESSRKAYDHNSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLMQPTQMSAQEDLGDDTGVIPTLNGDDRHKIVNVDQRQYGDVFKGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTTLFDELELDPPEIEPGVLKVLRTEKQYLGVYIWNMRGSDGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAQCLSHMNSGCTFTSPHLAQRVASTVYQNCEHADNYTAYCLGISHMEPSFGLILHDGGTTLKFVDTPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITPVNPGTSPLLRYAWTGGLAA VVLLCLVIFLICTA gi|46981496|gb|MKRIQINLILTIACIQLSTESQPTPVSITELYTSAATRKPDP 48 AAT07772.1|AVAPTSAATRKPDPAVAPTSAATRKPDPAVAPTSAATRK membranePDPAVAPTSAATRKPDPAVAPTSAATRKPDPAVAPTSAA glycoprotein CSRKPDPAVAPTSAASRKPDPAVAPTSAASRKPDPAANTQ [HumanHSQPPFLYENIQCVHGGIQSIPYFHTFIMPCYMRLTTGQQ herpesvirus 3]AAFKQQQKTYEQYSLDPEGSNITRWKSLIRPDLHIEVWFTRHLIDPHRQLGNALIRMPDLPVMLYSNSADLNLINNPEIFTHAKENYVIPDVKTTSDFSVTILSMDATTEGTYIWRVVNTKTKNVISEHSITVTTYYRPNITVVGDPVLTGQTYAAYCNVSKYYPPHSVRVRWTSRFGNIGKNFITDAIQEYANGLFSYVSAVRIPQQKQMDYPPPAIQCNVLWIRDGVSNMKYSAVVTPDVYPFPNVSIGIIDGHIVCTAKCVPRGVVHFVWWVNDSPINHENSEITGVCDQNKRFVNMQSSCPTSELDGPITYSCHLDGYPKKFPPFSAVYTYDASTYATTFSVVAVIIG VISILGTLGLIAVIATLCIRCCSgi|9625934|ref| MASHKWLLQIVFLKTITIAYCLHLQDDTPLFFGAKPLSD 49 NP_040182.1|VSLIITEPCVSSVYEAWDYAAPPVSNLSEALSGIVVKTKC envelopePVPEVILWFKDKQMAYWTNPYVTLKGLAQSVGEEHKS glycoprotein LGDIRDALLDALSGVWVDSTPSSTNIPENGCVWGADRLFQ [Human RVCQ herpesvirus 3]gi|9625925|ref| MGTQKKGPRSEKVSPYDTTTPEVEALDHQMDTLNWRI 50 NP_040172.1|WIIQVMMFTLGAVMLLATLIAASSEYTGIPCFYAAVVDY envelopeELFNATLDGGVWSGNRGGYSAPVLFLEPHSVVAFTYYT glycoprotein MALTAMAMAVYTLITAAIIHRETKNQRVRQSSGVAWLVV [HumanDPTTLFWGLLSLWLLNAVVLLLAYKQIGVAATLYLGHF herpesvirus 3]ATSVIFTTYFCGRGKLDETNIKAVANLRQQSVFLYRLAGPTRAVFVNLMAALMAICILFVSLMLELVVANHLHTGLWSSVSVAMSTFSTLSVVYLIVSELILAHYIHVLIGPSLGTLVACATLGTAAHSYMDRLYDPISVQSPRLIPTTRGTLACLAVFSVVMLLLRLMRAYVYHRQKRSRFYGAVRRVPERVRGYIRKVKPAHRNSRRTNYPSQGYGYVYENDSTYETDRE DELLYERSNSGWE gi|9625912|ref|MFALVLAVVILPLWTTANKSYVTPTPATRSIGHMSALLR 51 NP_040160.1|EYSDRNMSLKLEAFYPTGFDEELIKSLHWGNDRKHVFL envelopeVIVKVNPTTHEGDVGLVIFPKYLLSPYHFKAEHRAPFPA glycoprotein HGRFGFLSHPVTPDVSFFDSSFAPYLTTQHLVAFTTFPPNPL [HumanVWHLERAETAATAERPFGVSLLPARPTVPKNTILEHKAH herpesvirus 3]FATWDALARHTFFSAEAIITNSTLRIHVPLFGSVWPIRYWATGSVLLTSDSGRVEVNIGVGFMSSLISLSSGPPIELIVVPHTVKLNAVTSDTTWFQLNPPGPDPGPSYRVYLLGRGLDMNFSKHATVDICAYPEESLDYRYHLSMAHTEALRMTTKADQHDINEESYYHIAARIATSIFALSEMGRTTEYFLLDEIVDVQYQLKFLNYILMRIGAGAHPNTISGTSDLIFADPSQLHDELSLLFGQVKPANVDYFISYDEARDQLKTAYALSRGQDHVNALSLARRVIMSIYKGLLVKQNLNATERQALFFASMILLNFREGLENSSRVLDGRTTLLLMTSMCTAAHATQAALNIQEGLAYLNPSKHMFTIPNVYSPCMGSLRTDLTEEIHVMNLLSAIPTRPGLNEVLHTQLDESEIFDAAFKTMMIFTTWTAKDLHILHTHVPEVFTCQDAAARNGEYVLILPAVQGHSYVITRNKPQRGLVYSLADVDVYNPISVVYLSRDTCVSEHGVIETVALPHPDNLKECLYCGSVFLRYLTTGAIMDIIIIDSKDTERQLAAMGNSTIPPFNPDMHGDDSKAVLLFPNGTVVTLLGFERRQAIRMSGQYLGASLGGAFLAVVGFGIIG WMLCGNSRLREYNKIPLTgi|584403829|gb| MFYEALKAELVYTRAVHGFRPRANCVVLSDYIPRVACN 52 AHB80298.1|MGTVNKPVVGVLMGFGIITGTLRITNPVRASVLRYDDFH envelopeIDEDKLDTNSVYEPYYHSDHAESSWVNRGESSRKAYDH glycoprotein ENSPYIWPRNDYDGFLENAHEHHGVYNQGRGIDSGERLM [HumanQPTQMSAQEDLGDDTGIHVIPTLNGDDRHKIVNVDQRQ herpesvirus 3]YGDVFKGDLNPKPQGQRLIEVSVEENHPFTLRAPIQRIYGVRYTETWSFLPSLTCTGDAAPAIQHICLKHTTCFQDVVVDVDCAENTKEDQLAEISYRFQGKKEADQPWIVVNTSTLFDELELDPPEIEPGVLKVLRTEKQYLGVYIWNMRGSDGTSTYATFLVTWKGDEKTRNPTPAVTPQPRGAEFHMWNYHSHVFSVGDTFSLAMHLQYKIHEAPFDLLLEWLYVPIDPTCQPMRLYSTCLYHPNAPQCLSHMNSGCTFTSPHLAQRVASTVYQNCEHADNYTAYCLGISHMEPSFGLILHDGGTTLKFVDTPESLSGLYVFVVYFNGHVEAVAYTVVSTVDHFVNAIEERGFPPTAGQPPATTKPKEITPVNPGTSPLLRYAAWTGGLAAVVLLCLVIFLICTAKRMRVKAYRVDKSPYNQSMYYAGLPVDDFEDSESTDTEEEFGNAIGGSHGGSSYTVY IDKTR gi|46981513|gb|MFVTAVVSVSPSSFYESLQVEPTQSEDITRSAHLGDGDEI 53 AAT07789.11REAIHKSQDAETKPTFYVCPPPTGSTIVRLEPTRTCPDYH glycoprotein BLGKNFTEGIAVVYKENIAAYKFKATVYYKDVIVSTAWA [HumanGSSYTQITNRYADRVPIPVSEITDTIDKFGKCSSKATYVR herpesvirus 3]NNHKVEAFNEDKNPQDMPLIASKYNSVGSKAWHTTNDTYMVAGTPGTYRTGTSVNCIIEEVEARSIFPYDSFGLSTGDIIYMSPFFGLRDGAYREHSNYAMDRFHQFEGYRQRDLDTRALLEPAARNFLVTPHLTVGWNWKPKRTEVCSLVKWREVEDVVRDEYAHNFRFTMKTLSTTFISETNEFNLNQIHLSQCVKEEARAIINRIYTTRYNSSHVRTGDIQTYLARGGFVVVFQPLLSNSLARLYLQELVRENTNHSPQKHPTRNTRSRRSVPVELRANRTITTTSSVEFAMLQFTYDHIQEHVNEMLARISSSWCQLQNRERALWSGLFPINPSALASTILDQRVKARILGDVISVSNCPELGSDTRIILQNSMRVSGSTTRCYSRPLISIVSLNGSGTVEGQLGTDNELIMSRDLLEPCVANHKRYFLFGHHYVYYEDYRYVREIAVHDVGMISTYVDLNLTLLKDREFMPLQVYTRDELRDTGLLDYSEIQRRNQMHSLRFYDIDKVVQYDSGTAIMQGMAQFFQGLGTAGQAVGHVVLGATGALLSTVHGFTTFLSNPFGALAVGLLVLAGLVAAFFAYRYVLKLKTSPMKALYPLTTKGLKQLPEGMDPFAEKPNATDTPIEEIGDSQNTEPSVNSGFDPDKFREAQEMIKYMTLVSAAERQESKARKKNKTSALLTSRLTGLALRNRRGYS RVRTENVTGV gi|46981487|gb|MQALGIKTEHFIIMCLLSGHAVFTLWYTARVKFEHECVY 54 AAT07763.1|ATTVINGGPVVWGSYNNSLIYVTFVNHSTFLDGLSGYDY glycoprotein KSCRENLLSGDTMVKTAISTPLHDKIRIVLGTRNCHAYFW [HumanCVQLKMIFFAWFVYGMYLQFRRIRRMFGPFRSSCELISPT herpesvirus 3]SYSLNYVTRVISNILLGYPYTKLARLLCDVSMRRDGMSKVFNADPISFLYMHKGVTLLMLLEVIAHISSGCIVLLTLGVAYTPCALLYPTYIRILAWVVVCTLAIVELISYVRPKPTKDNHLNHINTGGIRGICTTCCATVMSGLAIKCFYIVIFAIAVV IFMHYEQRVQVSLFGESENSQKHgi|443500633|gb| MGSITASFILITMQILFFCEDSSGEPNFAERNFWHASCSAR 55AGC94499.1| GVYIDGSMITTLFFYASLLGVCVALISLAYHACFRLFTRS glycoprotein NVLRSTW [Human herpesvirus 3] Ig heavy chain MDWTWILFLVAAATRVHS 56epsilon-1 signal peptide (IgE HC SP) IgGk chain V-IIIMETPAQLLFLLLLWLPDTTG 57 region HAH signal peptide (IgGk SP) JapaneseMLGSNSGQRVVFTILLLLVAPAYS 109 encephalitis PRM signal sequenceVSVg protein MKCLLYLAFLFIGVNCA 110 signal sequence JapaneseMWLVSLAIVTACAGA 111 encephalitis JEV signal sequence

TABLE 16 Flagellin Nucleic Acid Sequences SEQ ID Name Sequence NO:NT (5′ TCAAGCTTTTGGACCCTCGTACAGAAGCTAATACGACTCACTAT 112 UTR, ORF,AGGGAAATAAGAGAGAAAAGAAGAGTAAGAAGAAATATAAG 3′ UTR)AGCCACCATGGCACAAGTCATTAATACAAACAGCCTGTCGCTGTTGACCCAGAATAACCTGAACAAATCCCAGTCCGCACTGGGCACTGCTATCGAGCGTTTGTCTTCCGGTCTGCGTATCAACAGCGCGAAAGACGATGCGGCAGGACAGGCGATTGCTAACCGTTTTACCGCGAACATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGCGCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCGAATGGTACTAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTGAACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAGGACAACACCCTGACCATCCAGGTTGGTGCCAACGACGGTGAAACTATCGATATTGATTTAAAAGAAATCAGCTCTAAAACACTGGGACTTGATAAGCTTAATGTCCAAGATGCCTACACCCCGAAAGAAACTGCTGTAACCGTTGATAAAACTACCTATAAAAATGGTACAGATCCTATTACAGCCCAGAGCAATACTGATATCCAAACTGCAATTGGCGGTGGTGCAACGGGGGTTACTGGGGCTGATATCAAATTTAAAGATGGTCAATACTATTTAGATGTTAAAGGCGGTGCTTCTGCTGGTGTTTATAAAGCCACTTATGATGAAACTACAAAGAAAGTTAATATTGATACGACTGATAAAACTCCGTTGGCAACTGCGGAAGCTACAGCTATTCGGGGAACGGCCACTATAACCCACAACCAAATTGCTGAAGTAACAAAAGAGGGTGTTGATACGACCACAGTTGCGGCTCAACTTGCTGCAGCAGGGGTTACTGGCGCCGATAAGGACAATACTAGCCTTGTAAAACTATCGTTTGAGGATAAAAACGGTAAGGTTATTGATGGTGGCTATGCAGTGAAAATGGGCGACGATTTCTATGCCGCTACATATGATGAGAAAACAGGTGCAATTACTGCTAAAACCACTACTTATACAGATGGTACTGGCGTTGCTCAAACTGGAGCTGTGAAATTTGGTGGCGCAAATGGTAAATCTGAAGTTGTTACTGCTACCGATGGTAAGACTTACTTAGCAAGCGACCTTGACAAACATAACTTCAGAACAGGCGGTGAGCTTAAAGAGGTTAATACAGATAAGACTGAAAACCCACTGCAGAAAATTGATGCTGCCTTGGCACAGGTTGATACACTTCGTTCTGACCTGGGTGCGGTTCAGAACCGTTTCAACTCCGCTATCACCAACCTGGGCAATACCGTAAATAACCTGTCTTCTGCCCGTAGCCGTATCGAAGATTCCGACTACGCAACCGAAGTCTCCAACATGTCTCGCGCGCAGATTCTGCAGCAGGCCGGTACCTCCGTTCTGGCGCAGGCGAACCAGGTTCCGCAAAACGTCCTCTCTTTACTGCGTTGATAATAGGCTGGAGCCTCGGTGGCCATGCTTCTTGCCCCTTGGGCCTCCCCCCAGCCCCTCCTCCCCTTCCTGCACCCGTACCCCCGTGGTCTTTGAATAAAGTCTGAGTGGGCGG C ORFATGGCACAAGTCATTAATACAAACAGCCTGTCGCTGTTGACCC 113 Sequence,AGAATAACCTGAACAAATCCCAGTCCGCACTGGGCACTGCTAT NTCGAGCGTTTGTCTTCCGGTCTGCGTATCAACAGCGCGAAAGACGATGCGGCAGGACAGGCGATTGCTAACCGTTTTACCGCGAACATCAAAGGTCTGACTCAGGCTTCCCGTAACGCTAACGACGGTATCTCCATTGCGCAGACCACTGAAGGCGCGCTGAACGAAATCAACAACAACCTGCAGCGTGTGCGTGAACTGGCGGTTCAGTCTGCGAATGGTACTAACTCCCAGTCTGACCTCGACTCCATCCAGGCTGAAATCACCCAGCGCCTGAACGAAATCGACCGTGTATCCGGCCAGACTCAGTTCAACGGCGTGAAAGTCCTGGCGCAGGACAACACCCTGACCATCCAGGTTGGTGCCAACGACGGTGAAACTATCGATATTGATTTAAAAGAAATCAGCTCTAAAACACTGGGACTTGATAAGCTTAATGTCCAAGATGCCTACACCCCGAAAGAAACTGCTGTAACCGTTGATAAAACTACCTATAAAAATGGTACAGATCCTATTACAGCCCAGAGCAATACTGATATCCAAACTGCAATTGGCGGTGGTGCAACGGGGGTTACTGGGGCTGATATCAAATTTAAAGATGGTCAATACTATTTAGATGTTAAAGGCGGTGCTTCTGCTGGTGTTTATAAAGCCACTTATGATGAAACTACAAAGAAAGTTAATATTGATACGACTGATAAAACTCCGTTGGCAACTGCGGAAGCTACAGCTATTCGGGGAACGGCCACTATAACCCACAACCAAATTGCTGAAGTAACAAAAGAGGGTGTTGATACGACCACAGTTGCGGCTCAACTTGCTGCAGCAGGGGTTACTGGCGCCGATAAGGACAATACTAGCCTTGTAAAACTATCGTTTGAGGATAAAAACGGTAAGGTTATTGATGGTGGCTATGCAGTGAAAATGGGCGACGATTTCTATGCCGCTACATATGATGAGAAAACAGGTGCAATTACTGCTAAAACCACTACTTATACAGATGGTACTGGCGTTGCTCAAACTGGAGCTGTGAAATTTGGTGGCGCAAATGGTAAATCTGAAGTTGTTACTGCTACCGATGGTAAGACTTACTTAGCAAGCGACCTTGACAAACATAACTTCAGAACAGGCGGTGAGCTTAAAGAGGTTAATACAGATAAGACTGAAAACCCACTGCAGAAAATTGATGCTGCCTTGGCACAGGTTGATACACTTCGTTCTGACCTGGGTGCGGTTCAGAACCGTTTCAACTCCGCTATCACCAACCTGGGCAATACCGTAAATAACCTGTCTTCTGCCCGTAGCCGTATCGAAGATTCCGACTACGCAACCGAAGTCTCCAACATGTCTCGCGCGCAGATTCTGCAGCAGGCCGGTACCTCCGTTCTGGCGCAGGCGAACCAGGTTCCGCAAAACGTCCTCTCTT TACTGCGT mRNAG*GGGAAAUAAGAGAGAAAAGAAGAGUAAGAAGAAAUAUAA 114 SequenceGAGCCACCAUGGCACAAGUCAUUAAUACAAACAGCCUGUCGC (assumesUGUUGACCCAGAAUAACCUGAACAAAUCCCAGUCCGCACUGG T100 tail)GCACUGCUAUCGAGCGUUUGUCUUCCGGUCUGCGUAUCAACAGCGCGAAAGACGAUGCGGCAGGACAGGCGAUUGCUAACCGUUUUACCGCGAACAUCAAAGGUCUGACUCAGGCUUCCCGUAACGCUAACGACGGUAUCUCCAUUGCGCAGACCACUGAAGGCGCGCUGAACGAAAUCAACAACAACCUGCAGCGUGUGCGUGAACUGGCGGUUCAGUCUGCGAAUGGUACUAACUCCCAGUCUGACCUCGACUCCAUCCAGGCUGAAAUCACCCAGCGCCUGAACGAAAUCGACCGUGUAUCCGGCCAGACUCAGUUCAACGGCGUGAAAGUCCUGGCGCAGGACAACACCCUGACCAUCCAGGUUGGUGCCAACGACGGUGAAACUAUCGAUAUUGAUUUAAAAGAAAUCAGCUCUAAAACACUGGGACUUGAUAAGCUUAAUGUCCAAGAUGCCUACACCCCGAAAGAAACUGCUGUAACCGUUGAUAAAACUACCUAUAAAAAUGGUACAGAUCCUAUUACAGCCCAGAGCAAUACUGAUAUCCAAACUGCAAUUGGCGGUGGUGCAACGGGGGUUACUGGGGCUGAUAUCAAAUUUAAAGAUGGUCAAUACUAUUUAGAUGUUAAAGGCGGUGCUUCUGCUGGUGUUUAUAAAGCCACUUAUGAUGAAACUACAAAGAAAGUUAAUAUUGAUACGACUGAUAAAACUCCGUUGGCAACUGCGGAAGCUACAGCUAUUCGGGGAACGGCCACUAUAACCCACAACCAAAUUGCUGAAGUAACAAAAGAGGGUGUUGAUACGACCACAGUUGCGGCUCAACUUGCUGCAGCAGGGGUUACUGGCGCCGAUAAGGACAAUACUAGCCUUGUAAAACUAUCGUUUGAGGAUAAAAACGGUAAGGUUAUUGAUGGUGGCUAUGCAGUGAAAAUGGGCGACGAUUUCUAUGCCGCUACAUAUGAUGAGAAAACAGGUGCAAUUACUGCUAAAACCACUACUUAUACAGAUGGUACUGGCGUUGCUCAAACUGGAGCUGUGAAAUUUGGUGGCGCAAAUGGUAAAUCUGAAGUUGUUACUGCUACCGAUGGUAAGACUUACUUAGCAAGCGACCUUGACAAACAUAACUUCAGAACAGGCGGUGAGCUUAAAGAGGUUAAUACAGAUAAGACUGAAAACCCACUGCAGAAAAUUGAUGCUGCCUUGGCACAGGUUGAUACACUUCGUUCUGACCUGGGUGCGGUUCAGAACCGUUUCAACUCCGCUAUCACCAACCUGGGCAAUACCGUAAAUAACCUGUCUUCUGCCCGUAGCCGUAUCGAAGAUUCCGACUACGCAACCGAAGUCUCCAACAUGUCUCGCGCGCAGAUUCUGCAGCAGGCCGGUACCUCCGUUCUGGCGCAGGCGAACCAGGUUCCGCAAAACGUCCUCUCUUUACUGCGUUGAUAAUAGGCUGGAGCCUCGGUGGCCAUGCUUCUUGCCCCUUGGGCCUCCCCCCAGCCCCUCCUCCCCUUCCUGCACCCGUACCCCCGUGGUCUUUGAAUAAAGUCUGAGUGGGCGGCAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAUCUAG

It should be understood that each of the ORF sequences provided hereinmay be combined with a 5′ and/or 3′ UTR, such as those described herein.It should also be understood that the 5′ and/or 3′ UTR for eachconstruct may be omitted, modified or substituted for a different UTRsequences in any one of the vaccines as provided herein.

TABLE 17 Flagellin Amino Acid Sequences SEQ ID Name Sequence NO: ORFMAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAA 115 Sequence,GQAIANRFTANIKGLTQASRNANDGISIAQTTEGALNEINNNLQRV AARELAVQSANGTNSQSDLDSIQAEITQRLNEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKEISSKTLGLDKLNVQDAYTPKETAVTVDKTTYKNGTDPITAQSNTDIQTAIGGGATGVTGADIKFKDGQYYLDVKGGASAGVYKATYDETTKKVNIDTTDKTPLATAEATAIRGTATITHNQIAEVTKEGVDTTTVAAQLAAAGVTGADKDNTSLVKLSFEDKNGKVIDGGYAVKMGDDFYAATYDEKTGAITAKTTTYTDGTGVAQTGAVKFGGANGKSEVVTATDGKTYLASDLDKHNFRTGGELKEVNTDKTENPLQKIDAALAQVDTLRSDLGAVQNRFNSAITNLGNTVNNLSSARSRIEDSDYATEVSNMSRAQILQQAGTSVLAQA NQVPQNVLSLLR Flagellin-MAQVINTNSLSLLTQNNLNKSQSALGTAIERLSSGLRINSAKDDAA 116 GS linker-GQAIANRFTANIKGLTQASRNANDGISIAQTTEGALNEINNNLQRV circumsporRELAVQSANSTNSQSDLDSIQAEITQRLNEIDRVSGQTQFNGVKVL ozoiteAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDTLNVQQKYKVSD proteinTAATVTGYADTTIALDNSTFKASATGLGGTDQKIDGDLKFDDTTGKYYAKVTVTGGTGKDGYYEVSVDKTNGEVTLAGGATSPLTGGLPATATEDVKNVQVANADLTEAKAALTAAGVTGTASVVKMSYTDNNGKTIDGGLAVKVGDDYYSATQNKDGSISINTTKYTADDGTSKTALNKLGGADGKTEVVSIGGKTYAASKAEGHNFKAQPDLAEAAATTTENPLQKIDAALAQVDTLRSDLGAVQNRFNSAITNLGNTVNNLTSARSRIEDSDYATEVSNMSRAQILQQAGTSVLAQANQVPQNVLSLLRGGGGSGGGGSMMAPDPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNKNNQGNGQGHNMPNDPNRNVDENANANNAVKNNNNEEPSDKHIEQYLKKIKNSISTEWSPCSVTCGNGIQVRIKPGSANKPKDELDYENDIEKKICKMEKCSSVFNVVNS Flagellin-MMAPDPNANPNANPNANPNANPNANPNANPNANPNANPNANPN 117 RPVTANPNANPNANPNANPNANPNANPNANPNANPNANPNANPNKNN linker-QGNGQGHNMPNDPNRNVDENANANNAVKNNNNEEPSDKHIEQY circumsporLKKIKNSISTEWSPCSVTCGNGIQVRIKPGSANKPKDELDYENDIEK ozoiteKICKMEKCSSVFNVVNSRPVT MAQVINTNSLSLLTQNNLNKSQSA proteinLGTAIERLSSGLRINSAKDDAAGQAIANRFTANIKGLTQASRNANDGISIAQTTEGALNEINNNLQRVRELAVQSANSTNSQSDLDSIQAEITQRLNEIDRVSGQTQFNGVKVLAQDNTLTIQVGANDGETIDIDLKQINSQTLGLDTLNVQQKYKVSDTAATVTGYADTTIALDNSTFKASATGLGGTDQKIDGDLKFDDTTGKYYAKVTVTGGTGKDGYYEVSVDKTNGEVTLAGGATSPLTGGLPATATEDVKNVQVANADLTEAKAALTAAGVTGTASVVKMSYTDNNGKTIDGGLAVKVGDDYYSATQNKDGSISINTTKYTADDGTSKTALNKLGGADGKTEVVSIGGKTYAASKAEGHNFKAQPDLAEAAATTTENPLQKIDAALAQVDTLRSDLGAVQNRFNSAITNLGNTVNNLTSARSRIEDSDYATEVSNMSRAQILQ QAGTSVLAQANQVPQNVLSLLR

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

All references, including patent documents, disclosed herein areincorporated by reference in their entirety.

What is claimed is:
 1. A vaccine comprising at least one RNApolynucleotide having an open reading frame encoding at least onevaricella zoster virus (VZV) antigenic polypeptide formulated in a lipidnanoparticle comprising at least one cationic lipid selected fromcompounds of Formula (I):

or a salt or isomer thereof, wherein: R₁ is selected from the groupconsisting of C₅₋₃₀ alkyl, C₅₋₂₀ alkenyl, —R*YR″, —YR″, and —R″M′R′; R₂and R₃ are independently selected from the group consisting of H, C₁₋₁₄alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃, togetherwith the atom to which they are attached, form a heterocycle orcarbocycle; R₄ is selected from the group consisting of a C₃₋₆carbocycle, —(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, —CQ(R)₂, andunsubstituted C₁₋₆ alkyl, where Q is selected from a carbocycle,heterocycle, —OR, —O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H,—CXH₂, —CN, —N(R)₂, —C(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)C(O)N(R)₂,—N(R)C(S)N(R)₂, —N(R)R₈, —O(CH₂)_(n)OR, —N(R)C(═NR₉)N(R)₂,—N(R)C(═CHR₉)N(R)₂, —OC(O)N(R)₂, —N(R)C(O)OR, —N(OR)C(O)R, —N(OR)S(O)₂R,—N(OR)C(O)OR, —N(OR)C(O)N(R)₂, —N(OR)C(S)N(R)₂, —N(OR)C(═NR₉)N(R)₂,—N(OR)C(═CHR₉)N(R)₂, —C(═NR₉)N(R)₂, —C(═NR₉)R, —C(O)N(R)OR, and—C(R)N(R)₂C(O)OR, and each n is independently selected from 1, 2, 3, 4,and 5; each R₅ is independently selected from the group consisting ofC₁₋₃ alkyl, C₂₋₃ alkenyl, and H; each R₆ is independently selected fromthe group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl, and H; M and M′ areindependently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—, —N(R′)C(O)—,—C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—, —S(O)₂—,—S—S—, an aryl group, and a heteroaryl group; R₇ is selected from thegroup consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl, and H; R₈ is selected fromthe group consisting of C₃₋₆ carbocycle and heterocycle; R₉ is selectedfrom the group consisting of H, CN, NO₂, C₁₋₆ alkyl, —OR, —S(O)₂R,—S(O)₂N(R)₂, C₂₋₆ alkenyl, C₃₋₆ carbocycle and heterocycle; each R isindependently selected from the group consisting of C₁₋₃ alkyl, C₂₋₃alkenyl, and H; each R′ is independently selected from the groupconsisting of C₁₋₁₈ alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H; each R″is independently selected from the group consisting of C₃₋₁₄ alkyl andC₃₋₁₄ alkenyl; each R* is independently selected from the groupconsisting of C₁₋₁₂ alkyl and C₂₋₁₂ alkenyl; each Y is independently aC₃₋₆ carbocycle; each X is independently selected from the groupconsisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9,10, 11, 12, and
 13. 2. The vaccine of claim 1, wherein a subset ofcompounds of Formula (I) includes those in which when R₄ is —(CH₂)_(n)Q,—(CH₂)_(n)CHQR, —CHQR, or —CQ(R)₂, then (i) Q is not —N(R)₂ when n is 1,2, 3, 4 or 5, or (ii) Q is not 5, 6, or 7-membered heterocycloalkyl whenn is 1 or
 2. 3. The vaccine of claim 1, wherein a subset of compounds ofFormula (I) includes those in which R₁ is selected from the groupconsisting of C₅₋₃₀ alkyl, C₅₋₂₀ alkenyl, —R*YR″, —YR″, and —R″M′R′; R₂and R₃ are independently selected from the group consisting of H, C₁₋₁₄alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃, togetherwith the atom to which they are attached, form a heterocycle orcarbocycle; R₄ is selected from the group consisting of a C₃₋₆carbocycle, —(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, —CQ(R)₂, andunsubstituted C₁₋₆ alkyl, where Q is selected from a C₃₋₆ carbocycle, a5- to 14-membered heteroaryl having one or more heteroatoms selectedfrom N, O, and S, —OR, —O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H,—CXH₂, —CN, —C(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)C(O)N(R)₂,—N(R)C(S)N(R)₂, —CRN(R)₂C(O)OR, —N(R)R₈, —O(CH₂)_(n)OR,—N(R)C(═NR₉)N(R)₂, —N(R)C(═CHR₉)N(R)₂, —OC(O)N(R)₂, —N(R)C(O)OR,—N(OR)C(O)R, —N(OR)S(O)₂R, —N(OR)C(O)OR, —N(OR)C(O)N(R)₂,—N(OR)C(S)N(R)₂, —N(OR)C(═NR₉)N(R)₂, —N(OR)C(═CHR₉)N(R)₂, —C(═NR₉)N(R)₂,—C(═NR₉)R, —C(O)N(R)OR, and a 5- to 14-membered heterocycloalkyl havingone or more heteroatoms selected from N, O, and S which is substitutedwith one or more substituents selected from oxo (═O), OH, amino, mono-or di-alkylamino, and C₁₋₃ alkyl, and each n is independently selectedfrom 1, 2, 3, 4, and 5; each R₅ is independently selected from the groupconsisting of C₁₋₃ alkyl, C₂₋₃ alkenyl, and H; each R₆ is independentlyselected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl, and H; Mand M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, —S—S—, an aryl group, and a heteroaryl group; R₇ is selectedfrom the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl, and H; R₈ isselected from the group consisting of C₃₋₆ carbocycle and heterocycle;R₉ is selected from the group consisting of H, CN, NO₂, C₁₋₆ alkyl, —OR,—S(O)₂R, —S(O)₂N(R)₂, C₂₋₆ alkenyl, C₃₋₆ carbocycle and heterocycle;each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H; each R′ is independently selected from thegroup consisting of C₁₋₁₈ alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl; each R* is independently selected from thegroup consisting of C₁₋₁₂ alkyl and C₂₋₁₂ alkenyl; each Y isindependently a C₃₋₆ carbocycle; each X is independently selected fromthe group consisting of F, Cl, Br, and I; and m is selected from 5, 6,7, 8, 9, 10, 11, 12, and 13, or salts or isomers thereof.
 4. The vaccineof claim 1, wherein a subset of compounds of Formula (I) includes thosein which R₁ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀alkenyl, —R*YR″, —YR″, and —R″M′R′; R₂ and R₃ are independently selectedfrom the group consisting of H, C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″,—YR″, and —R*OR″, or R₂ and R₃, together with the atom to which they areattached, form a heterocycle or carbocycle; R₄ is selected from thegroup consisting of a C₃₋₆ carbocycle, —(CH₂)_(n)Q, —(CH₂)_(n)CHQR,—CHQR, —CQ(R)₂, and unsubstituted C₁₋₆ alkyl, where Q is selected from aC₃₋₆ carbocycle, a 5- to 14-membered heterocycle having one or moreheteroatoms selected from N, O, and S, —OR, —O(CH₂)_(n)N(R)₂, —C(O)OR,—OC(O)R, —CX₃, —CX₂H, —CXH₂, —CN, —C(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R,—N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂, —CRN(R)₂C(O)OR, —N(R)R₈, —O(CH₂)_(n)OR,—N(R)C(═NR₉)N(R)₂, —N(R)C(═CHR₉)N(R)₂, —OC(O)N(R)₂, —N(R)C(O)OR,—N(OR)C(O)R, —N(OR)S(O)₂R, —N(OR)C(O)OR, —N(OR)C(O)N(R)₂,—N(OR)C(S)N(R)₂, —N(OR)C(═NR₉)N(R)₂, —N(OR)C(═CHR₉)N(R)₂, —C(═NR₉)R,—C(O)N(R)OR, and —C(═NR₉)N(R)₂, and each n is independently selectedfrom 1, 2, 3, 4, and 5; and when Q is a 5- to 14-membered heterocycleand (i) R₄ is —(CH₂)_(n)Q in which n is 1 or 2, or (ii) R₄ is—(CH₂)_(n)CHQR in which n is 1, or (iii) R₄ is —CHQR, and —CQ(R)₂, thenQ is either a 5- to 14-membered heteroaryl or 8- to 14-memberedheterocycloalkyl; each R₅ is independently selected from the groupconsisting of C₁₋₃ alkyl, C₂₋₃ alkenyl, and H; each R₆ is independentlyselected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl, and H; Mand M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, —S—S—, an aryl group, and a heteroaryl group; R₇ is selectedfrom the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl, and H; R₈ isselected from the group consisting of C₃₋₆ carbocycle and heterocycle;R₉ is selected from the group consisting of H, CN, NO₂, C₁₋₆ alkyl, —OR,—S(O)₂R, —S(O)₂N(R)₂, C₂₋₆ alkenyl, C₃₋₆ carbocycle and heterocycle;each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H; each R′ is independently selected from thegroup consisting of C₁₋₁₈ alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl; each R* is independently selected from thegroup consisting of C₁₋₁₂ alkyl and C₂₋₁₂ alkenyl; each Y isindependently a C₃₋₆ carbocycle; each X is independently selected fromthe group consisting of F, Cl, Br, and I; and m is selected from 5, 6,7, 8, 9, 10, 11, 12, and 13, or salts or isomers thereof.
 5. The vaccineof claim 1, wherein a subset of compounds of Formula (I) includes thosein which R₁ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀alkenyl, —R*YR″, —YR″, and —R″M′R′; R₂ and R₃ are independently selectedfrom the group consisting of H, C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″,—YR″, and —R*OR″, or R₂ and R₃, together with the atom to which they areattached, form a heterocycle or carbocycle; R₄ is selected from thegroup consisting of a C₃₋₆ carbocycle, —(CH₂)_(n)Q, —(CH₂)_(n)CHQR,—CHQR, —CQ(R)₂, and unsubstituted C₁₋₆ alkyl, where Q is selected from aC₃₋₆ carbocycle, a 5- to 14-membered heteroaryl having one or moreheteroatoms selected from N, O, and S, —OR, —O(CH₂)_(n)N(R)₂, —C(O)OR,—OC(O)R, —CX₃, —CX₂H, —CXH₂, —CN, —C(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R,—N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂, —CRN(R)₂C(O)OR, —N(R)R₈, —O(CH₂)_(n)OR,—N(R)C(═NR₉)N(R)₂, —N(R)C(═CHR₉)N(R)₂, —OC(O)N(R)₂, —N(R)C(O)OR,—N(OR)C(O)R, —N(OR)S(O)₂R, —N(OR)C(O)OR, —N(OR)C(O)N(R)₂,—N(OR)C(S)N(R)₂, —N(OR)C(═NR₉)N(R)₂, —N(OR)C(═CHR₉)N(R)₂, —C(═NR₉)R,—C(O)N(R)OR, and —C(═NR₉) N(R)₂, and each n is independently selectedfrom 1, 2, 3, 4, and 5; each R₅ is independently selected from the groupconsisting of C₁₋₃ alkyl, C₂₋₃ alkenyl, and H; each R₆ is independentlyselected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl, and H; Mand M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, —S—S—, an aryl group, and a heteroaryl group; R₇ is selectedfrom the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl, and H; R₈ isselected from the group consisting of C₃₋₆ carbocycle and heterocycle;R₉ is selected from the group consisting of H, CN, NO₂, C₁₋₆ alkyl, —OR,—S(O)₂R, —S(O)₂N(R)₂, C₂₋₆ alkenyl, C₃₋₆ carbocycle and heterocycle;each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H; each R′ is independently selected from thegroup consisting of C₁₋₁₈ alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl; each R* is independently selected from thegroup consisting of C₁₋₁₂ alkyl and C₂₋₁₂ alkenyl; each Y isindependently a C₃₋₆ carbocycle; each X is independently selected fromthe group consisting of F, Cl, Br, and I; and m is selected from 5, 6,7, 8, 9, 10, 11, 12, and 13, or salts or isomers thereof.
 6. The vaccineof claim 1, wherein subset of compounds of Formula (I) includes those inwhich R₁ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀alkenyl, —R*YR″, —YR″, and —R″M′R′; R₂ and R₃ are independently selectedfrom the group consisting of H, C₂₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″,—YR″, and —R*OR″, or R₂ and R₃, together with the atom to which they areattached, form a heterocycle or carbocycle; R₄ is —(CH₂)_(n)Q or—(CH₂)_(n)CHQR, where Q is —N(R)₂, and n is selected from 3, 4, and 5;each R₅ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H; each R₆ is independently selected from thegroup consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl, and H; M and M′ areindependently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—, —N(R′)C(O)—,—C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—, —S(O)₂—,—S—S—, an aryl group, and a heteroaryl group; R₇ is selected from thegroup consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl, and H; each R isindependently selected from the group consisting of C₁₋₃ alkyl, C₂₋₃alkenyl, and H; each R′ is independently selected from the groupconsisting of C₁₋₁₈ alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H; each R″is independently selected from the group consisting of C₃₋₁₄ alkyl andC₃₋₁₄ alkenyl; each R* is independently selected from the groupconsisting of C₁₋₁₂ alkyl and C₁₋₁₂ alkenyl; each Y is independently aC₃₋₆ carbocycle; each X is independently selected from the groupconsisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9,10, 11, 12, and 13, or salts or isomers thereof.
 7. The vaccine of claim1, wherein a subset of compounds of Formula (I) includes those in whichR₁ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀ alkenyl,—R*YR″, —YR″, and —R″M′R′; R₂ and R₃ are independently selected from thegroup consisting of C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and—R*OR″, or R₂ and R₃, together with the atom to which they are attached,form a heterocycle or carbocycle; R₄ is selected from the groupconsisting of —(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, and —CQ(R)₂, where Qis —N(R)₂, and n is selected from 1, 2, 3, 4, and 5; each R₅ isindependently selected from the group consisting of Ca-3 alkyl, C₂₋₃alkenyl, and H; each R₆ is independently selected from the groupconsisting of Ca-3 alkyl, C₂₋₃ alkenyl, and H; M and M′ areindependently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—, —N(R′)C(O)—,—C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—, —S(O)₂—,—S—S—, an aryl group, and a heteroaryl group; R₇ is selected from thegroup consisting of Ca-3 alkyl, C₂₋₃ alkenyl, and H; each R isindependently selected from the group consisting of C₁₋₃ alkyl, C₂₋₃alkenyl, and H; each R′ is independently selected from the groupconsisting of C₁₋₁₈ alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H; each R″is independently selected from the group consisting of C₃₋₁₄ alkyl andC₃₋₁₄ alkenyl; each R* is independently selected from the groupconsisting of C₁₋₂ alkyl and C₁₋₂ alkenyl; each Y is independently aC₃₋₆ carbocycle; each X is independently selected from the groupconsisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9,10, 11, 12, and 13, or salts or isomers thereof.
 8. The vaccine of claim1, wherein a subset of compounds of Formula (I) includes those ofFormula (IA):

or a salt or isomer thereof, wherein l is selected from 1, 2, 3, 4, and5; m is selected from 5, 6, 7, 8, and 9; M₁ is a bond or M′; R₄ isunsubstituted C₁₋₃ alkyl, or —(CH₂)_(n)Q, in which Q is OH,—NHC(S)N(R)₂, —NHC(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)R₈,—NHC(═NR₉)N(R)₂, —NHC(═CHR₉)N(R)₂, —OC(O)N(R)₂, —N(R)C(O)OR, heteroarylor heterocycloalkyl; M and M′ are independently selected from —C(O)O—,—OC(O)—, —C(O)N(R′)—, —P(O)(OR′)O—, —S—S—, an aryl group, and aheteroaryl group; and R₂ and R₃ are independently selected from thegroup consisting of H, C₁₋₁₄ alkyl, and C₂₋₁₄ alkenyl.
 9. The vaccine ofany one of claims 1-8, wherein the at least one RNA polynucleotide hasan open reading frame encoding two or more VZV antigenic polypeptides.10. The vaccine of any one of claims 1-9, wherein the at least one VZVantigenic polypeptide is a VZV glycoprotein.
 11. The vaccine of any oneof claims 1-9, wherein the at least one VZV antigenic polypeptide is aVZV tegument protein.
 12. The vaccine of claim 10, wherein the VZVglycoprotein is selected from VZV gE, gI, gB, gH, gK, gL, gC, gN, andgM.
 13. The vaccine of claim 12, wherein the VZV glycoprotein is VZV gE.14. The vaccine of claim 12, wherein the VZV glycoprotein is VZV gI. 15.The vaccine of claim 13, wherein the VZV glycoprotein is a variant VZVgE polypeptide.
 16. The vaccine of claim 15, wherein the variant VZV gEpolypeptide is a truncated polypeptide lacking the anchor domain (ERretention domain).
 17. The vaccine of claim 15, wherein the variant VZVgE polypeptide is a truncated polypeptide lacking the carboxy terminaltail domain.
 18. The vaccine of claim 16, wherein the VZV antigenicpolypeptide is a truncated VZV gE polypeptide comprising amino acids1-561 of SEQ ID NO:
 10. 19. The vaccine of claim 17, wherein the VZVantigenic polypeptide is a VZV gE variant polypeptide comprising aminoacids 1-573 of SEQ ID NO:
 18. 20. The vaccine of claim 15, wherein thevariant VZV gE polypeptide has at least one mutation in at least onemotif associated with ER retention, endocytosis, and/or localization tothe golgi or trans-golgi network.
 21. The vaccine of claim 20, whereinthe variant VZV gE polypeptide has at least one mutation in at least onephosphorylated acidic motif(s).
 22. The vaccine of claim 21, wherein thevariant VZV gE polypeptide has a Y582G mutation with respect to SEQ IDNO:
 10. 23. The vaccine of claim 21, wherein the variant VZV gEpolypeptide has a Y569A mutation with respect to SEQ ID NO:
 10. 24. Thevaccine of claim 21, wherein the variant VZV gE polypeptide has a Y582Gmutation and a Y569A mutation with respect to SEQ ID NO:
 10. 25. Thevaccine of claim 19, wherein the VZV antigenic polypeptide has a Y569Amutation with respect to SEQ ID NO:
 10. 26. The vaccine of claim 13, orany of claims 20-24, wherein the variant VZV gE polypeptide has anA-E-A-A-D-A sequence (SEQ ID NO: 58).
 27. The vaccine of claim 13 or anyof claims 20-24, wherein the variant VZV gE polypeptide has an IgKappasequence at the C-terminus.
 28. The vaccine of claim 18 or 19, whereinthe VZV gE antigenic polypeptide has an IgKappa sequence at theC-terminus.
 29. The vaccine of claim 12, wherein the at least one RNApolynucleotide encodes VZV gE and gI antigenic polypeptides.
 30. Thevaccine of claim 13 or any of claims 15-28, wherein the vaccinecomprises at least one RNA polynucleotide having an open reading frameencoding a gI antigenic polypeptide.
 31. The vaccine of claim 13 or anyof claims 15-28, wherein the vaccine comprises at least one RNApolynucleotide having an open reading frame encoding an antigenicpolypeptide selected from VZV gB, gH, gK, gL, gC, gN, and gM.
 32. Thevaccine of claim 11, wherein at least one RNA polynucleotide has an openreading frame encoding a VZV antigenic polypeptide selected from VZV gE,gI, gB, gH, gK, gL, gC, gN, and gM.
 33. The vaccine of any of claims12-32, further comprising a live attenuated VZV, whole inactivated VZV,or VZV VLP.
 34. The vaccine of any one of claims 1-10, wherein the atleast one RNA polynucleotide is encoded by at least one nucleic acidsequence selected from SEQ ID NO: 1-8, 37, 39, 41, 60, 62, 64, 66, 68,70, 72, 74, 76, 78, 80, 82, 84, 86, 88, or 90, or wherein the at leastone RNA polynucleotide is encoded by at least one nucleic acid sequenceselected from SEQ ID NO: 6, 37, 39, 84, or
 86. 35. The vaccine of anyone of claims 1-10, wherein the at least one RNA polynucleotidecomprises a sequence selected from SEQ ID NO: 63, 67, 71, 75, 79, 83,87, 91, 92-108, 123-131, 134, and 142-150.
 36. The vaccine of any one ofclaims 1-10, wherein the at least one RNA polynucleotide comprises atleast one fragment of a nucleic acid sequence or at least one epitope ofa mRNA sequence selected from SEQ ID NO: 63, 67, 71, 75, 79, 83, 87, 91,92-108, 123-131, 134, and 142-150.
 37. The vaccine of claim 13, whereinthe at least one RNA polynucleotide is encoded by SEQ ID NO:
 1. 38. Thevaccine of claim 14, wherein the at least one RNA polynucleotide isencoded by SEQ ID NO:
 2. 39. The vaccine of claim 18, wherein the atleast one RNA polynucleotide is encoded by SEQ ID NO:
 3. 40. The vaccineof claim 19, wherein the at least one RNA polynucleotide is encoded bySEQ ID NO:
 5. 41. The vaccine of claim 25, wherein the at least one RNApolynucleotide is encoded by SEQ ID NO: 6, 37, 39, 84, or
 86. 42. Thevaccine of claim 26, wherein the at least one RNA polynucleotide isencoded by SEQ ID NO:
 7. 43. The vaccine of claim 26, wherein the atleast one RNA polynucleotide is encoded by SEQ ID NO:
 8. 44. The vaccineof claim 10, wherein the at least one RNA polynucleotide encodes anantigenic polypeptide having at least 90% identity to the amino acidsequence of any one of SEQ ID NO: 10, 14, 26, 30, 42 and 45-55.
 45. Thevaccine of claim 10, wherein the at least one RNA polynucleotide encodesan antigenic polypeptide having at least 95% identity to the amino acidsequence of any one of SEQ ID NO: 10, 14, 26, 30, 38, 42 and 45-55. 46.The vaccine of claim 10, wherein the at least one RNA polynucleotideencodes an antigenic polypeptide having at least 96% identity to theamino acid sequence of any one of SEQ ID NO: 10, 14, 26, 30, 38, 42 and45-55.
 47. The vaccine of claim 10, wherein the at least one RNApolynucleotide encodes an antigenic polypeptide having at least 97%identity to the amino acid sequence of any one of SEQ ID NO: 10, 14, 26,30, 38, 42 and 45-55.
 48. The vaccine of claim 10, wherein the at leastone RNA polynucleotide encodes an antigenic polypeptide having at least98% identity to the amino acid sequence of any one of SEQ ID NO: 10, 14,26, 30, 38, 42 and 45-55.
 49. The vaccine of claim 10, wherein the atleast one RNA polynucleotide encodes an antigenic polypeptide having atleast 99% identity to the amino acid sequence of any one of SEQ ID NO:10, 14, 26, 30, 38, 42 and 45-55.
 50. The vaccine of any one of claims1-49, wherein the open reading frame from which the polynucleotide isencoded is codon-optimized.
 51. The vaccine of any one of claims 1-50,wherein the vaccine is multivalent.
 52. The vaccine of any one of claims1-51, wherein the RNA polynucleotide comprises at least one chemicalmodification.
 53. The vaccine of claim 52, further comprising a secondchemical modification.
 54. The vaccine of claim 52 or 53, wherein thechemical modification is selected from the group consisting ofpseudouridine, N1-methylpseudouridine, N1-ethylpseudouridine,2-thiouridine, 4′-thiouridine, 5-methylcytosine,2-thio-1-methyl-1-deaza-pseudouridine, 2-thio-1-methyl-pseudouridine,2-thio-5-aza-uridine, 2-thio-dihydropseudouridine,2-thio-dihydrouridine, 2-thio-pseudouridine,4-methoxy-2-thio-pseudouridine, 4-methoxy-pseudouridine,4-thio-1-methyl-pseudouridine, 4-thio-pseudouridine, 5-aza-uridine,dihydropseudouridine, 5-methoxyuridine, and 2′-O-methyl uridine.
 55. Thevaccine of claim 52, wherein 80% of the uracil in the open reading framehave a chemical modification.
 56. The vaccine of claim 55, wherein 100%of the uracil in the open reading frame have a chemical modification.57. The vaccine of any one of claims 52-56, wherein the chemicalmodification is in the 5-position of the uracil.
 58. The vaccine ofclaim 57, wherein the chemical modification is N1-methylpseudouridine.59. The vaccine of any one of claims 1-58, wherein the vaccine isformulated within a cationic lipid nanoparticle.
 60. The vaccine ofclaim 59, wherein the lipid nanoparticle further comprises aPEG-modified lipid, a sterol and a non-cationic lipid.
 61. The vaccineof claim 60, wherein the non-cationic lipid is a neutral lipid.
 62. Thevaccine of claim 60 or 61, wherein the sterol is a cholesterol.
 63. Thevaccine of any one of claims 1-62, wherein the lipid nanoparticle has amolar ratio of about 20-60% cationic lipid, about 5-25% non-cationiclipid, about 25-55% sterol, and about 0.5-15% PEG-modified lipid. 64.The vaccine of any one of claims 1-63, wherein the lipid nanoparticlehas a polydispersity value of less than 0.4.
 65. The vaccine of any oneof claims 1-64, wherein the lipid nanoparticle has a net neutral chargeat a neutral pH.
 66. The vaccine of any one of claims 1-65, wherein thenanoparticle has a mean diameter of 50-200 nm.
 67. A VZV vaccine of anyone of claims 1-66 for use in a method of inducing an antigen specificimmune response in a subject, the method comprising administering thevaccine to the subject in an effective amount to produce an antigenspecific immune response.
 68. Use of a VZV vaccine of any one of claims1-66 in the manufacture of a medicament for use in a method of inducingan antigen specific immune response in a subject, the method comprisingadministering the vaccine to the subject in an effective amount toproduce an antigen specific immune response.
 69. A VZV vaccinecomprising at least one ribonucleic acid (RNA) polynucleotide, said RNApolynucleotide having (i) a 5′ terminal cap, (ii) an open reading frameencoding at least one VZV antigenic polypeptide, and (iii) a 3′ polyAtail formulated in a lipid nanoparticle comprising at least one cationiclipid selected from compounds of Formula (I):

or a salt or isomer thereof, wherein: R₁ is selected from the groupconsisting of C₅₋₃₀ alkyl, C₅₋₂₀ alkenyl, —R*YR″, —YR″, and —R″M′R′; R₂and R₃ are independently selected from the group consisting of H, C₁₋₁₄alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃, togetherwith the atom to which they are attached, form a heterocycle orcarbocycle; R₄ is selected from the group consisting of a C₃₋₆carbocycle, —(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, —CQ(R)₂, andunsubstituted C₁₋₆ alkyl, where Q is selected from a carbocycle,heterocycle, —OR, —O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H,—CXH₂, —CN, —N(R)₂, —C(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)C(O)N(R)₂,—N(R)C(S)N(R)₂, —N(R)R₈, —O(CH₂)_(n)OR, —N(R)C(═NR₉)N(R)₂,—N(R)C(═CHR₉)N(R)₂, —OC(O)N(R)₂, —N(R)C(O)OR, —N(OR)C(O)R, —N(OR)S(O)₂R,—N(OR)C(O)OR, —N(OR)C(O)N(R)₂, —N(OR)C(S)N(R)₂, —N(OR)C(═NR₉)N(R)₂,—N(OR)C(═CHR₉)N(R)₂, —C(═NR₉)N(R)₂, —C(═NR₉)R, —C(O)N(R)OR, and—C(R)N(R)₂C(O)OR, and each n is independently selected from 1, 2, 3, 4,and 5; each R₅ is independently selected from the group consisting ofC₁₋₃ alkyl, C₂₋₃ alkenyl, and H; each R₆ is independently selected fromthe group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl, and H; M and M′ areindependently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—, —N(R′)C(O)—,—C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—, —S(O)₂—,—S—S—, an aryl group, and a heteroaryl group; R₇ is selected from thegroup consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl, and H; R₈ is selected fromthe group consisting of C₃₋₆ carbocycle and heterocycle; R₉ is selectedfrom the group consisting of H, CN, NO₂, C₁₋₆ alkyl, —OR, —S(O)₂R,—S(O)₂N(R)₂, C₂₋₆ alkenyl, C₃₋₆ carbocycle and heterocycle; each R isindependently selected from the group consisting of C₁₋₃ alkyl, C₂₋₃alkenyl, and H; each R′ is independently selected from the groupconsisting of C₁₋₁₈ alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H; each R″is independently selected from the group consisting of C₃₋₁₄ alkyl andC₃₋₁₄ alkenyl; each R* is independently selected from the groupconsisting of C₁₋₁₂ alkyl and C₂₋₁₂ alkenyl; each Y is independently aC₃₋₆ carbocycle; each X is independently selected from the groupconsisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9,10, 11, 12, and
 13. 70. The vaccine of claim 69, wherein a subset ofcompounds of Formula (I) includes those in which when R₄ is —(CH₂)_(n)Q,—(CH₂)_(n)CHQR, —CHQR, or —CQ(R)₂, then (i) Q is not —N(R)₂ when n is 1,2, 3, 4 or 5, or (ii) Q is not 5, 6, or 7-membered heterocycloalkyl whenn is 1 or
 2. 71. The vaccine of claim 69, wherein a subset of compoundsof Formula (I) includes those in which R₁ is selected from the groupconsisting of C₅₋₃₀ alkyl, C₅₋₂₀ alkenyl, —R*YR″, —YR″, and —R″M′R′; R₂and R₃ are independently selected from the group consisting of H, C₁₋₁₄alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃, togetherwith the atom to which they are attached, form a heterocycle orcarbocycle; R₄ is selected from the group consisting of a C₃₋₆carbocycle, —(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, —CQ(R)₂, andunsubstituted C₁₋₆ alkyl, where Q is selected from a C₃₋₆ carbocycle, a5- to 14-membered heteroaryl having one or more heteroatoms selectedfrom N, O, and S, —OR, —O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H,—CXH₂, —CN, —C(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)C(O)N(R)₂,—N(R)C(S)N(R)₂, —CRN(R)₂C(O)OR, —N(R)R₈, —O(CH₂)_(n)OR,—N(R)C(═NR₉)N(R)₂, —N(R)C(═CHR₉)N(R)₂, —OC(O)N(R)₂, —N(R)C(O)OR,—N(OR)C(O)R, —N(OR)S(O)₂R, —N(OR)C(O)OR, —N(OR)C(O)N(R)₂,—N(OR)C(S)N(R)₂, —N(OR)C(═NR₉)N(R)₂, —N(OR)C(═CHR₉)N(R)₂, —C(═NR₉)N(R)₂,—C(═NR₉)R, —C(O)N(R)OR, and a 5- to 14-membered heterocycloalkyl havingone or more heteroatoms selected from N, O, and S which is substitutedwith one or more substituents selected from oxo (═O), OH, amino, mono-or di-alkylamino, and C₁₋₃ alkyl, and each n is independently selectedfrom 1, 2, 3, 4, and 5; each R₅ is independently selected from the groupconsisting of C₁₋₃ alkyl, C₂₋₃ alkenyl, and H; each R₆ is independentlyselected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl, and H; Mand M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, —S—S—, an aryl group, and a heteroaryl group; R₇ is selectedfrom the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl, and H; R₈ isselected from the group consisting of C₃₋₆ carbocycle and heterocycle;R₉ is selected from the group consisting of H, CN, NO₂, C₁₋₆ alkyl, —OR,—S(O)₂R, —S(O)₂N(R)₂, C₂₋₆ alkenyl, C₃₋₆ carbocycle and heterocycle;each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H; each R′ is independently selected from thegroup consisting of C₁₋₁₈ alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H;each R″ is independently selected from the group consisting of C₃₋₁₄alkyl and C₃₋₁₄ alkenyl; each R* is independently selected from thegroup consisting of C₁₋₁₂ alkyl and C₂₋₁₂ alkenyl; each Y isindependently a C₃₋₆ carbocycle; each X is independently selected fromthe group consisting of F, Cl, Br, and I; and m is selected from 5, 6,7, 8, 9, 10, 11, 12, and 13, or salts or isomers thereof.
 72. Thevaccine of claim 69, wherein a subset of compounds of Formula (I)includes those in which R₁ is selected from the group consisting ofC₅₋₃₀ alkyl, C₅₋₂₀ alkenyl, —R*YR″, —YR″, and —R″M′R′; R₂ and R₃ areindependently selected from the group consisting of H, C₁₋₁₄ alkyl,C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ and R₃, together with theatom to which they are attached, form a heterocycle or carbocycle; R₄ isselected from the group consisting of a C₃₋₆ carbocycle, —(CH₂)_(n)Q,—(CH₂)_(n)CHQR, —CHQR, —CQ(R)₂, and unsubstituted C₁₋₆ alkyl, where Q isselected from a C₃₋₆ carbocycle, a 5- to 14-membered heterocycle havingone or more heteroatoms selected from N, O, and S, —OR,—O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H, —CXH₂, —CN, —C(O)N(R)₂,—N(R)C(O)R, —N(R)S(O)₂R, —N(R)C(O)N(R)₂, —N(R)C(S)N(R)₂, —CRN(R)₂C(O)OR,—N(R)R₈, —O(CH₂)_(n)OR, —N(R)C(═NR₉)N(R)₂, —N(R)C(═CHR₉)N(R)₂,—OC(O)N(R)₂, —N(R)C(O)OR, —N(OR)C(O)R, —N(OR)S(O)₂R, —N(OR)C(O)OR,—N(OR)C(O)N(R)₂, —N(OR)C(S)N(R)₂, —N(OR)C(═NR₉)N(R)₂,—N(OR)C(═CHR₉)N(R)₂, —C(═NR₉)R, —C(O)N(R)OR, and —C(═NR₉)N(R)₂, and eachn is independently selected from 1, 2, 3, 4, and 5; and when Q is a 5-to 14-membered heterocycle and (i) R₄ is —(CH₂)_(n)Q in which n is 1 or2, or (ii) R₄ is —(CH₂)_(n)CHQR in which n is 1, or (iii) R₄ is —CHQR,and —CQ(R)₂, then Q is either a 5- to 14-membered heteroaryl or 8- to14-membered heterocycloalkyl; each R₅ is independently selected from thegroup consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl, and H; each R₆ isindependently selected from the group consisting of C₁₋₃ alkyl, C₂₋₃alkenyl, and H; M and M′ are independently selected from —C(O)O—,—OC(O)—, —C(O)N(R′)—, —N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—,—CH(OH)—, —P(O)(OR′)O—, —S(O)₂—, —S—S—, an aryl group, and a heteroarylgroup; R₇ is selected from the group consisting of C₁₋₃ alkyl, C₂₋₃alkenyl, and H; R₈ is selected from the group consisting of C₃₋₆carbocycle and heterocycle; R₉ is selected from the group consisting ofH, CN, NO₂, C₁₋₆ alkyl, —OR, —S(O)₂R, —S(O)₂N(R)₂, C₂₋₆ alkenyl, C₃₋₆carbocycle and heterocycle; each R is independently selected from thegroup consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl, and H; each R′ isindependently selected from the group consisting of C₁₋₈ alkyl, C₂₋₁₈alkenyl, —R*YR″, —YR″, and H; each R″ is independently selected from thegroup consisting of C₃₋₁₄ alkyl and C₃₋₁₄ alkenyl; each R* isindependently selected from the group consisting of C₁₋₁₂ alkyl andC₂₋₁₂ alkenyl; each Y is independently a C₃₋₆ carbocycle; each X isindependently selected from the group consisting of F, Cl, Br, and I;and m is selected from 5, 6, 7, 8, 9, 10, 11, 12, and 13, or salts orisomers thereof.
 73. The vaccine of claim 69, wherein a subset ofcompounds of Formula (I) includes those in which R₁ is selected from thegroup consisting of C₅₋₃₀ alkyl, C₅₋₂₀ alkenyl, —R*YR″, —YR″, and—R″M′R′; R₂ and R₃ are independently selected from the group consistingof H, C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″, and —R*OR″, or R₂ andR₃, together with the atom to which they are attached, form aheterocycle or carbocycle; R₄ is selected from the group consisting of aC₃₋₆ carbocycle, —(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, —CQ(R)₂, andunsubstituted C₁₋₆ alkyl, where Q is selected from a C₃₋₆ carbocycle, a5- to 14-membered heteroaryl having one or more heteroatoms selectedfrom N, O, and S, —OR, —O(CH₂)_(n)N(R)₂, —C(O)OR, —OC(O)R, —CX₃, —CX₂H,—CXH₂, —CN, —C(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)C(O)N(R)₂,—N(R)C(S)N(R)₂, —CRN(R)₂C(O)OR, —N(R)R₈, —O(CH₂)_(n)OR,—N(R)C(═NR₉)N(R)₂, —N(R)C(═CHR₉)N(R)₂, —OC(O)N(R)₂, —N(R)C(O)OR,—N(OR)C(O)R, —N(OR)S(O)₂R, —N(OR)C(O)OR, —N(OR)C(O)N(R)₂,—N(OR)C(S)N(R)₂, —N(OR)C(═NR₉)N(R)₂, —N(OR)C(═CHR₉)N(R)₂, —C(═NR₉)R,—C(O)N(R)OR, and —C(═NR₉)N(R)₂, and each n is independently selectedfrom 1, 2, 3, 4, and 5; each R₅ is independently selected from the groupconsisting of C₁₋₃ alkyl, C₂₋₃ alkenyl, and H; each R₆ is independentlyselected from the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl, and H; Mand M′ are independently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—,—N(R′)C(O)—, —C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—,—S(O)₂—, —S—S—, an aryl group, and a heteroaryl group; R₇ is selectedfrom the group consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl, and H; R₈ isselected from the group consisting of C₃₋₆ carbocycle and heterocycle;R₉ is selected from the group consisting of H, CN, NO₂, C₁₋₆ alkyl, —OR,—S(O)₂R, —S(O)₂N(R)₂, C₂₋₆ alkenyl, C₃₋₆ carbocycle and heterocycle;each R is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H; each R′ is independently selected from thegroup consisting of C₁₋₈ alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H; eachR″ is independently selected from the group consisting of C₃₋₁₄ alkyland C₃₋₁₄ alkenyl; each R* is independently selected from the groupconsisting of C₁₋₁₂ alkyl and C₂₋₁₂ alkenyl; each Y is independently aC₃₋₆ carbocycle; each X is independently selected from the groupconsisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9,10, 11, 12, and 13, or salts or isomers thereof.
 74. The vaccine ofclaim 69, wherein subset of compounds of Formula (I) includes those inwhich R₁ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀alkenyl, —R*YR″, —YR″, and —R″M′R′; R₂ and R₃ are independently selectedfrom the group consisting of H, C₂₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″,—YR″, and —R*OR″, or R₂ and R₃, together with the atom to which they areattached, form a heterocycle or carbocycle; R₄ is —(CH₂)_(n)Q or—(CH₂)_(n)CHQR, where Q is —N(R)₂, and n is selected from 3, 4, and 5;each R₅ is independently selected from the group consisting of C₁₋₃alkyl, C₂₋₃ alkenyl, and H; each R₆ is independently selected from thegroup consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl, and H; M and M′ areindependently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—, —N(R′)C(O)—,—C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—, —S(O)₂—,—S—S—, an aryl group, and a heteroaryl group; R₇ is selected from thegroup consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl, and H; each R isindependently selected from the group consisting of C₁₋₃ alkyl, C₂₋₃alkenyl, and H; each R′ is independently selected from the groupconsisting of C₁₋₁₈ alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H; each R″is independently selected from the group consisting of C₃₋₁₄ alkyl andC₃₋₁₄ alkenyl; each R* is independently selected from the groupconsisting of C₁₋₁₂ alkyl and C₁₋₁₂ alkenyl; each Y is independently aC₃₋₆ carbocycle; each X is independently selected from the groupconsisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9,10, 11, 12, and 13, or salts or isomers thereof.
 75. The vaccine ofclaim 69, wherein a subset of compounds of Formula (I) includes those inwhich R₁ is selected from the group consisting of C₅₋₃₀ alkyl, C₅₋₂₀alkenyl, —R*YR″, —YR″, and —R″M′R′; R₂ and R₃ are independently selectedfrom the group consisting of C₁₋₁₄ alkyl, C₂₋₁₄ alkenyl, —R*YR″, —YR″,and —R*OR″, or R₂ and R₃, together with the atom to which they areattached, form a heterocycle or carbocycle; R₄ is selected from thegroup consisting of —(CH₂)_(n)Q, —(CH₂)_(n)CHQR, —CHQR, and —CQ(R)₂,where Q is —N(R)₂, and n is selected from 1, 2, 3, 4, and 5; each R₅ isindependently selected from the group consisting of C₁₋₃ alkyl, C₂₋₃alkenyl, and H; each R₆ is independently selected from the groupconsisting of C₁₋₃ alkyl, C₂₋₃ alkenyl, and H; M and M′ areindependently selected from —C(O)O—, —OC(O)—, —C(O)N(R′)—, —N(R′)C(O)—,—C(O)—, —C(S)—, —C(S)S—, —SC(S)—, —CH(OH)—, —P(O)(OR′)O—, —S(O)₂—,—S—S—, an aryl group, and a heteroaryl group; R₇ is selected from thegroup consisting of C₁₋₃ alkyl, C₂₋₃ alkenyl, and H; each R isindependently selected from the group consisting of C₁₋₃ alkyl, C₂₋₃alkenyl, and H; each R′ is independently selected from the groupconsisting of C₁₋₁₈ alkyl, C₂₋₁₈ alkenyl, —R*YR″, —YR″, and H; each R″is independently selected from the group consisting of C₃₋₁₄ alkyl andC₃₋₁₄ alkenyl; each R* is independently selected from the groupconsisting of C₁₋₁₂ alkyl and C₁₋₁₂ alkenyl; each Y is independently aC₃₋₆ carbocycle; each X is independently selected from the groupconsisting of F, Cl, Br, and I; and m is selected from 5, 6, 7, 8, 9,10, 11, 12, and 13, or salts or isomers thereof.
 76. The vaccine ofclaim 69, wherein a subset of compounds of Formula (I) includes those ofFormula (IA):

or a salt or isomer thereof, wherein l is selected from 1, 2, 3, 4, and5; m is selected from 5, 6, 7, 8, and 9; M₁ is a bond or M′; R₄ isunsubstituted C₁₋₃ alkyl, or —(CH₂)_(n)Q, in which Q is OH,—NHC(S)N(R)₂, —NHC(O)N(R)₂, —N(R)C(O)R, —N(R)S(O)₂R, —N(R)R₈,—NHC(═NR₉)N(R)₂, —NHC(═CHR₉)N(R)₂, —OC(O)N(R)₂, —N(R)C(O)OR, heteroarylor heterocycloalkyl; M and M′ are independently selected from —C(O)O—,—OC(O)—, —C(O)N(R′)—, —P(O)(OR′)O—, —S—S—, an aryl group, and aheteroaryl group; and R₂ and R₃ are independently selected from thegroup consisting of H, C₁₋₁₄ alkyl, and C₂₋₁₄ alkenyl.
 77. The vaccineof any one of claims 69-76, wherein the at least one mRNA polynucleotideis encoded by a sequence comprising the sequence identified by SEQ IDNO:
 6. 78. The vaccine of any one of claims 69-76, wherein the at leastone mRNA polynucleotide is encoded by a sequence comprising the sequenceidentified by SEQ ID NO:
 37. 79. The vaccine of any one of claims 69-76,wherein the at least one mRNA polynucleotide is encoded by a sequencecomprising the sequence identified by SEQ ID NO:
 39. 80. The vaccine ofany one of claims 69-76, wherein the at least one mRNA polynucleotide isencoded by a sequence comprising the sequence identified by SEQ ID NO:84.
 81. The vaccine of any one of claims 69-76, wherein the at least onemRNA polynucleotide is encoded by a sequence comprising the sequenceidentified by SEQ ID NO:
 86. 82. The vaccine of any one of claims 69-76,wherein the at least one mRNA polynucleotide comprises a sequenceidentified by SEQ ID NO:
 128. 83. The vaccine of any one of claims69-76, wherein the at least one mRNA polynucleotide comprises a sequenceidentified by SEQ ID NO: 99 or
 133. 84. The vaccine of any one of claims69-76, wherein the at least one mRNA polynucleotide comprises a sequenceidentified by SEQ ID NO: 107 or
 134. 85. The vaccine of any one ofclaims 69-76, wherein the at least one VZV antigenic polypeptidecomprises a sequence identified by SEQ ID NO:
 38. 86. The vaccine of anyone of claims 69-85, wherein the 5′ terminal cap is or comprises7mG(5′)ppp(5′)NlmpNp.
 87. The vaccine of any one of claims 69-86comprising at least one messenger ribonucleic acid (mRNA) polynucleotidecomprising a nucleic acid sequence identified by any one of SEQ ID NO:142-150 having (i) a 5′ terminal cap, (ii) an open reading frameencoding a VZV antigenic polypeptide, and (iii) a 3′ polyA tail, wherein5′ terminal cap is or comprises 7mG(5′)ppp(5′)NlmpNp.
 88. The vaccine ofany one of claims 69-87, wherein 100% of the uracil in the open readingframe is modified to include N1-methyl pseudouridine at the 5-positionof the uracil.
 89. A method of preventing or treating VZV infectioncomprising administering to a subject the vaccine of any of claims 1-67or 69-88.
 90. A method of inducing an antigen-specific immune responsein a subject, the method comprising administering to the subject thevaccine of any one of claims 1-67 or 69-89 in an amount effective toproduce an antigen-specific immune response in the subject.
 91. Themethod of claim 90, wherein the antigen specific immune responsecomprises a T cell response or a B cell response.
 92. The method ofclaim 90 or 91, wherein the subject is administered a single dose of thevaccine.
 93. The method of claim 90 or 91, wherein the subject isadministered a first dose and a second (booster) dose of the vaccine.94. The method of any one of claims 90-93, wherein the vaccine isadministered to the subject by intradermal injection or intramuscularinjection.
 95. The method of any one of claims 90-94, wherein ananti-antigenic polypeptide antibody titer produced in the subject isincreased by at least 1 log relative to a control.
 96. The method of anyone of claims 90-95, wherein an anti-antigenic polypeptide antibodytiter produced in the subject is increased by 1-3 log relative to acontrol.
 97. The method of any one of claims 90-96, wherein theanti-antigenic polypeptide antibody titer produced in the subject isincreased at least 2 times relative to a control.
 98. The method of anyone of claims 90-97, wherein the anti-antigenic polypeptide antibodytiter produced in the subject is increased 2-10 times relative to acontrol.
 99. The method of any one of claims 95-98, wherein the controlis an anti-antigenic polypeptide antibody titer produced in a subjectwho has not been administered a vaccine against the virus.
 100. Themethod of any one of claims 95-98, wherein the control is ananti-antigenic polypeptide antibody titer produced in a subject who hasbeen administered a live attenuated vaccine or an inactivated vaccineagainst the virus.
 101. The method of any one of claims 95-98, whereinthe control is an anti-antigenic polypeptide antibody titer produced ina subject who has been administered a recombinant protein vaccine orpurified protein vaccine against the virus.
 102. The method of any oneof claims 95-98, wherein the control is an anti-antigenic polypeptideantibody titer produced in a subject who has been administered a VLPvaccine against the virus.
 103. The method of any one of claims 90-102,wherein the effective amount is a dose equivalent to an at least 2-foldreduction in the standard of care dose of a recombinant protein vaccineor a purified protein vaccine against the virus, and wherein ananti-antigenic polypeptide antibody titer produced in the subject isequivalent to an anti-antigenic polypeptide antibody titer produced in acontrol subject administered the standard of care dose of a recombinantprotein vaccine or a purified protein vaccine against the virus,respectively.
 104. The method of any one of claims 90-102, wherein theeffective amount is a dose equivalent to an at least 2-fold reduction inthe standard of care dose of a live attenuated vaccine or an inactivatedvaccine against the virus, and wherein an anti-antigenic polypeptideantibody titer produced in the subject is equivalent to ananti-antigenic polypeptide antibody titer produced in a control subjectadministered the standard of care dose of a live attenuated vaccine oran inactivated vaccine against the virus, respectively.
 105. The methodof any one of claims 90-102, wherein the effective amount is a doseequivalent to an at least 2-fold reduction in the standard of care doseof a VLP vaccine against the virus, and wherein an anti-antigenicpolypeptide antibody titer produced in the subject is equivalent to ananti-antigenic polypeptide antibody titer produced in a control subjectadministered the standard of care dose of a VLP vaccine against thevirus.
 106. The method of any one of claims 90-105, wherein theeffective amount is a total dose of 50 μg-1000 g.
 107. The method ofclaim 105, wherein the effective amount is a dose of 25 μg, 100 μg, 400μg, or 500 μg administered to the subject a total of two times.
 108. Themethod of any one of claims 90-107, wherein the efficacy of the vaccineagainst the virus is greater than 65%.
 109. The method of any one ofclaims 90-108, wherein the vaccine immunizes the subject against thevirus for up to 2 years.
 110. The method of any one of claims 90-108,wherein the vaccine immunizes the subject against the virus for morethan 2 years.
 111. The method of any one of claims 90-110, wherein thesubject has an age of about 1 year old to about 10 years old.
 112. Themethod of any one of claims 90-111, wherein the subject has been exposedto the virus, wherein the subject is infected with the virus, or whereinthe subject is at risk of infection by the virus.
 113. The method of anyone of claims 90-112, wherein the subject is immunocompromised.
 114. Apharmaceutical composition for use in vaccination of a subjectcomprising an effective dose of the vaccine of any one of claims 1-67 or69-88, wherein the effective dose is sufficient to produce detectablelevels of antigen as measured in serum of the subject at 1-72 hours postadministration.
 115. The composition of claim 114, wherein the cut offindex of the antigen is 1-2.
 116. A pharmaceutical composition for usein vaccination of a subject comprising an effective dose of the vaccineof any one of claims 1-67 or 69-88, wherein the effective dose issufficient to produce a 1,000-10,000 neutralization titer produced byneutralizing antibody against said antigen as measured in serum of thesubject at 1-72 hours post administration.